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. 2004 Mar;135(3):391–397. doi: 10.1111/j.1365-2249.2004.02399.x

Evidence that factors other than particular thyrotropin receptor T cell epitopes contribute to the development of hyperthyroidism in murine Graves' disease

P N PICHURIN *, CHUN-RONG CHEN *, Y NAGAYAMA †, O PICHURINA *, B RAPOPORT *, S M MCLACHLAN *
PMCID: PMC1808963  PMID: 15008970

Abstract

Immunization with thyrotropin receptor (TSHR)-adenovirus is an effective approach for inducing thyroid stimulating antibodies and Graves’ hyperthyroidism in BALB/c mice. In contrast, mice of the same strain vaccinated with TSHR-DNA have low or absent TSHR antibodies and their T cells recognize restricted epitopes on the TSHR. In the present study, we tested the hypothesis that immunization with TSHR-adenovirus induces a wider, or different, spectrum of TSHR T cell epitopes in BALB/c mice. Because TSHR antibody levels rose progressively from one to three TSHR-adenovirus injections, we compared T cell responses from mice immunized once or three times. Mice in the latter group were subdivided into animals that developed hyperthyroidism and those that remained euthyroid. Unexpectedly, splenocytes from mice immunized once, as well as splenocytes from hyperthyroid and euthyroid mice (three injections), all produced interferon-γ in response to the same three synthetic peptides (amino acid residues 52–71, 67–86 and 157–176). These peptides were also the major epitopes recognized by TSHR-DNA plasmid vaccinated mice. We observed lesser responses to a wide range of additional peptides in mice injected three times with TSHR-adenovirus, but the pattern was more consistent with increased background ‘noise’ than with spreading from primary epitopes to dominant secondary epitopes. In conclusion, these data suggest that factors other than particular TSHR T cell epitopes (such as adenovirus-induced expression of conformationally intact TSHR protein), contribute to the generation of thyroid stimulating antibodies with consequent hyperthyroidism in TSHR-adenovirus immunized mice.

Keywords: T cell epitopes, interferon-γ, tumour necrosis factor-α, linear antibody epitopes, thyrotropin-receptor, murine Graves’ disease

INTRODUCTION

Immunizing mice with DNA encoding the TSHR in a replication deficient adenovirus is an effective approach for inducing Graves’ disease in BALB/c mice [1–3]. Mice of this strain also develop hyperthyroidism after multiple injections of TSHR-expressing B cells [4] or dendritic cells [5], confirming that the BALB/c strain is genetically susceptible to experimentally induced Graves’ disease. In contrast, BALB/c mice vaccinated with TSHR-DNA in a plasmid do not develop hyperthyroidism [6–8]. In one study, TSHR antibodies developed in virtually all TSHR-DNA vaccinated BALB/c mice [6]. In contrast, we and others observed low or undetectable levels of antibodies generated by vaccination with TSHR-plasmid [1,7–9]. Overall therefore the lack of hyperthyroidism in this strain is likely to be related, at least in part, to low TSHR antibody levels induced by TSHR-DNA plasmid vaccination.

Antibody production by B lymphocytes requires ‘help’ from T lymphocytes in the form of cytokines. Previously, to understand why TSHR-DNA vaccination is relatively ineffective at inducing TSHR antibodies, we investigated the T cell response to TSHR antigen. Challenging spleen cells from animals injected with TSHR-DNA with TSHR antigen in vitro induced responses measured by cell proliferation (thymidine incorporation) and production of several T helper-1 (Th1)-cytokines, including interferon-gamma (IFN-γ) and, at lower levels, interleukin-2 (IL-2) and tumour necrosis factor (TNF-α) [7,9,10]. No Th2 cytokines (such as IL-4 or IL-5) were detected. Because earlier studies had suggested that Th2 responses were characteristic features of Graves’ disease models (for example [11], we attempted to enhance antibody responses by immune deviation away from Th1. However, TSHR antibody levels were not enhanced in IFN-γ knock-out mice or by employing an IFN-γ ‘decoy’ together with TSHR-DNA [9].

Recently, we used a panel of synthetic overlapping peptides, corresponding to the amino acid sequence of the TSHR, to analyse T cell responses in TSHR-DNA immunized mice. Spleen cells from BALB/c and NOD.H-2h4 mice responded to different peptides, as would be expected from their different Major Histocompatibility Complex (MHC) genes [10]. However, an unexpected finding was the restricted number of peptides recognized by mice of either strain. In particular, two groups of nonoverlapping peptides were recognized by all BALB/c mice [10]. A cardinal feature of induced immunity is spreading of T cell epitopes as the immune response progresses. Such spreading can involve recognition of new epitopes on the same antigen or even intermolecular spreading to other antigens (for example [12–14]. In contrast to TSHR-DNA vaccinated mice, there are no data on TSHR T cell epitopes in the much more effective Graves’ disease model induced by TSHR-adenovirus immunization. In the present study we tested the hypothesis that, in the same strain of mice (BALB/c), TSHR-adenovirus immunization would induce a wider, or different, spectrum of TSHR T cell epitopes, hence accounting for the very high incidence of TSHR antibodies with functional activity leading to hyperthyroidism.

METHODS AND MATERIALS

Immunization of mice with TSHR-adenovirus

Generation, amplification and purification of recombinant adenovirus expressing human TSHR (AdCMVTSHR) were performed as previously described [1]. Female BALB/c mice (6–7 weeks; Jackson Laboratories, Bar Harbor, Maine, USA) were injected intramuscularly once or three times at 3 week intervals with 50 µl PBS containing 1011 particles of TSHR-adenovirus (Ad-TSHR) or control adenovirus expressing β-galactosidase (Ad-Con). Mice were euthanized at the following times: 1 week after a single injection of adenovirus, or 8 weeks after the third injection; in addition, blood was drawn from the tail vein one week after the second injection. The mice immunized three times with TSHR adenovirus were previously characterized for TSHR antibodies and hyperthyroidism [3]. 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 in a pathogen-free facility.

ELISA for TSHR antibodies

Sera were examined for recognition of TSHR-289, a variant of the receptor expressed in eukaryotic cells that corresponds approximately to the extracellular A-subunit recognized by human autoantibodies [15]. The protein was affinity purified from culture medium as described [16]. ELISA wells were coated with TSHR-289 (1 µg/ml) and exposed to mouse sera (diluted 1 : 102 and 1 : 103). Antibody binding was detected with antimouse IgG coupled to horse-radish peroxidase (Sigma Chemical Co., St. Louis, MO, USA). The signal was developed with o-phenylenediamine and H2O2 and optical density (OD) read at 490 nm.

TSHR peptides

The panel of 26 peptides spanning the extracellular domain of the TSHR extracellular domain of the TSHR has been described previously [17]. Peptides were synthesized using an automated 431 A peptide synthesizer, HPLC purified and their structures confirmed by mass spectrometry. Each peptide was 20 amino acids in length and overlapped the subsequent peptide by 5 amino acids. Peptides corresponding to residues 22–41, 37–56, 42–71, etc. (numbering includes the TSHR signal peptide, residues 1–21 that is cleaved from the mature TSHR protein) were designated A, B, C, etc. Three extracellular loop peptides corresponding to residues 471–494, 661–571 and 650–660 were designated EC1, EC2 and EC3, respectively. For use in vitro, peptides were resuspended in sterile distilled water and used at a final concentration of 10 µg/ml.

Response to TSHR antigen and peptides

Splenocytes (duplicate 200 µl aliquots of approximately 5 × 105 cells) were incubated in round bottomed 96 -well plates in the presence or absence of TSHR-antigen (TSHR 289, see above; 10 µg/ml), TSHR peptides (10 µg/ml) or Concanavalin A (Con 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, 50 µmβ-mercaptoethanol and 100 units/ml penicillin. After 5–6 days (37°C, 5% CO2), culture supernatants were centrifuged to remove cell debris and stored at − 80°C.

All culture supernatants were assayed for IFN-γ by ELISA (100 µl; in duplicate) using capture and biotinylated detection-antibodies from BD Pharmingen (San Diego, CA, USA). In addition, selected supernatants were analysed with the Cytometric Bead Array (CBA; BD Pharmingen). This kit contains fluorescent capture beads coated with antibodies specific for five murine cytokines, IFN-γ, IL-2, TNF-α, IL-4 and IL-5. Each cytokine-specific bead has a discrete fluorescent intensity detectable in the FL3 channel of a flow cytometer. Supernatants (50 µl) were incubated with the cytokine bead pool, followed by phycoerythrin (PE)-conjugated secondary antibodies. After washing, the intensity of PE binding was detected in the FL2 channel of a FACScan (Becton Dickinson, San Jose, CA, USA). Cytokine data are reported as pg/ml or ng/ml extrapolated from recombinant cytokine standard curves.

RESULTS

TSHR antibodies after TSHR-adenovirus immunization

TSHR antibodies (IgG class) were undetectable in BALB/c mice 7 days after one TSHR-adenovirus injection, as was the case in control-adenovirus immunized mice (Fig. 1). In contrast, after two injections of TSHR-adenovirus, TSHR antibodies were readily detectable and the levels increased significantly after three injections (Rank Sum test, P < 0·005).

Fig. 1.

Fig. 1

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TSHR antibody levels rise progressively in BALB/c mice injected once, twice or three times with TSHR-adenovirus (TSHR-Ad). Antibody levels were measured by ELISA in sera (1 : 102 and 1 : 103 dilution) obtained a week after a single injection (1×), a week after two injections (2×) and 8 weeks after three injections (3×) of TSHR-adenovirus. Data are reported as the mean +SEM values for optical density (OD) at 490 nm for 3 mice injected once, 10 mice injected twice and 8 mice injected three times. Also included are data for mice injected three times with control adenovirus (5 mice). The data for 8 mice after 3 injections (1 : 103 dilution) have been reported previously [3]. The dashed line indicates the mean +2SD for control mice (n = 5). Values significantly different for sera from mice immunized twice versus three time with TSHR adenovirus: *P = 0·007 (1 : 102 dilution); **P = 0·005 (1 : 103 dilution) (Rank Sum test).

IFN-γ response of splenocytes to TSHR antigen

The absence of antibodies after a single TSHR adenovirus injection versus high titres after three TSHR-adenovirus injections provided an opportunity to examine T cell recognition of the TSHR at the two extremes of the antibody response. We studied mice from three groups of mice immunized in the following manner: three injections with control adenovirus; one injection with TSHR-adenovirus; and three injections with TSHR-adenovirus. The third set was subdivided into two groups; one that did, and one that did not, develop Graves’-like hyperthyroidism, despite the presence of high titres of TSHR antibodies. These mice had previously been characterized for thyroid function and TSHR antibodies [3] but their splenocytes had not been studied.

We first assessed splenocyte responses after challenge with TSHR antigen by measuring IFN-γ production, as reported previously for TSHR-DNA vaccination [7,9,10] and more recently for TSHR-adenovirus immunization [18]. Despite the absence of TSHR antibodies after a single TSHR-adenovirus injection (Fig. 1), splenocytes produced IFN-γ when cultured in the presence but not in the absence of TSHR antigen (Fig. 2). Splenocytes from mice immunized three times with TSHR-adenovirus secreted some IFN-γ when cultured in medium alone (spontaneous secretion) and responded vigorously to TSHR antigen (paired t-test, P < 0·001). There was no difference in the magnitude of the IFN-γ response between mice that developed hyperthyroidism and the animals that remained euthyroid. No IFN-γ was produced by splenocytes from control-adenovirus immunized mice, whether cultured with or without TSHR antigen. However, splenocytes from all mice (control- or TSHR-adenovirus immunized) produced IFN-γ in response to mitogen, Concanavalin A.

Fig. 2.

Fig. 2

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IFN-γ response of splenocytes to TSHR antigen in culture. Splenocytes from BALB/c mice immunized once (1×; n = 3 mice) or three times with TSHR-adenovirus (3×; n = 11 mice), as well for control-adenovirus immunized mice (n = 4 mice), were incubated in the absence and presence of TSHR- antigen (TSHR-Ag, 10 µg/ml) or Concanavalin A (Con-A). After 6 days, supernatants were analysed for IFN-γ measured by ELISA. Data (pg/ml) are shown for individual euthyroid mice (○) and hyperthyroid mice (•). Values significantly different for splenocytes cultured with versus without TSHR-Ag: *P < 0·005; **P < 0·001, paired t-tests); values significantly different compared with splenocytes from control-adenovirus immunized mice incubated in medium only, †P = 0·022, Rank Sum test.

Other cytokine responses to TSHR antigen

The cytometric bead array was used to quantify IFN-γ, IL-2, TNF-α, IL-4 and IL-5 in culture supernatants from representative mice in each group. The data for IFN-γ were comparable to those obtained by ELISA (not shown) but the findings for TNF-α and IL-5 were novel. Unlike IFN-γ (Fig. 2), TNF-α was induced by TSHR antigen in splenocytes from all groups of mice, including control-adenovirus immunized animals (Fig. 3a). The mean TNF-α levels were higher for TSHR-antigen stimulated splenocytes from mice immunized three times with TSHR-adenovirus than from control-adenovirus immunized mice, 340 ± 46 versus 234 ± 26 pg/ml, respectively, but the difference was not statistically significant.

Fig. 3.

Fig. 3

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Production of (a) TNF-α and (b) IL-5 by splenocytes from BALB/c mice immunized with TSHR-adenovirus. Splenocytes from mice that received one (1×) or three (3×) injections of TSHR-adenovirus, or 3 injections of control-adenovirus, were incubated in the absence and presence of TSHR-antigen (TSHR-Ag, 10 µg/ml) or Concanavalin A (Con-A). After 6 days, supernatants were analysed for cytokine production by cytometric bead array (see Methods). Data are shown for individual mice as pg/ml TNF-α or IL-5. • Hyperthyroid, ○ Euthyroid. Values significantly higher for splenocytes cultured with versus without TSHR-Ag: *P < 0·03; **P < 0·001 (paired t-tests).

IL-5 was elevated in Con-A stimulated cultures and, unexpectedly, in TSHR-antigen stimulated splenocytes from one TSHR-adenovirus immunized mouse (Fig. 3b). Production of IL-5 by this particular animal, but not by other mice studied, was associated with secretion of large amounts of IFN-γ (>5500 pg/ml). Of the remaining two cytokines measured by the cytometric bead array, neither IL-2 nor IL-4 was induced by TSHR-antigen although both cytokines were induced by Con-A (116·2 ± 36·6 pg/ml of IL-2 and 62·7 ± 21·5 pg/ml IL-4; mean ± SEM, n = 4).

T cell epitopes recognized by TSHR-adenovirus immunized mice

Specific induction of IFN-γ following TSHR antigen challenge (Fig. 2) made it feasible to analyse the T cell epitopes involved in this response. IFN-γ production was measured by ELISA in splenocyte cultures from individual mice incubated with a panel of 29 synthetic peptides encompassing the TSHR ectodomain and the 3 extracellular loops. Peptides (20-mers) overlapped by five amino acid residues. Splenocytes from control-adenovirus injected mice were unresponsive to all peptides (Fig. 4a). In contrast, splenocytes from mice immunized once with TSHR-adenovirus responded to peptides C, D and J (Fig. 4b). Although production of IFN-γ was variable (note wide error bars), the magnitude of the peptide response was comparable with the TSHR-antigen response.

Fig. 4.

Fig. 4

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TSHR peptides recognized by BALB/c mice immunized with TSHR-adenovirus. Splenocytes were incubated for 6 days in medium with 29 TSHR peptides (10 µg/ml), in medium alone, or with TSHR antigen (TSHR-Ag). Supernatants were analysed for IFN-γ production by ELISA. The mean +SEM ng/ml IFN-γ-values are shown for the following groups: (a) Mice injected with control-adenovirus (n = 3); (b) Mice euthyroid after one TSHR-Ad injection (n = 3); (c) Mice euthyroid after 3 injections of TSHR-Ad (n = 4); (d) Mice hyperthyroid after 3 injections of TSHR-Ad (n = 6). The dominant peptides recognized are indicated in capital letters; *significantly different from medium only, peptides C, D and J, paired t-tests (P < 0·05).

Turning to the mice immunized three times with TSHR-adenovirus, the data are depicted separately for euthyroid mice versus hyperthyroid mice. Splenocytes from 4 mice that remained euthyroid responded to peptides C, D and J (Fig. 4c). Maximal responses to these peptides, were lower than for splenocytes from mice immunized once with TSHR-adenovirus (Fig. 4b) but were comparable with responses induced to TSHR-antigen. In addition, a low amplitude and variable response to numerous other peptides was observed. This ‘noise’ in the euthyroid mice immunized three times with TSHR-adenovirus was greater than in mice injected only once. Splenocytes from hyperthyroid animals had the highest noise levels. Nevertheless, in these hyperthyoid mice, only splenocyte responses to peptides C, D and J were significantly increased compared with splenocytes cultured in medium (Fig. 4d).

DISCUSSION

The present study was performed to determine the TSHR T cell epitopes recognized by BALB/c mice that develop Graves’-like hyperthyroidism following injection with TSHR-adenovirus. For comparison, we investigated TSHR epitopes recognized by similarly immunized mice that remained euthyroid despite the development of TSHR antibodies. Unexpectedly, we found that splenocytes from hyperthyroid and euthyroid mice responded to the same three major peptides from the panel of 29 overlapping synthetic peptides encompassing the TSHR ectodomain and the three extracellular loops. These peptides were recognized by spleen lymphocytes from mice that were TSHR antibody negative after a single TSHR-adenovirus injection and were also the major epitopes recognized by TSHR-DNA plasmid vaccinated mice [10]. Consequently, no T cell epitope appears to be associated specifically with the development of thyroid stimulating antibodies (TSAb), the cause of hyperthyroidism in Graves’ disease.

All three peptides recognized by splenocytes from BALB/c mice immunized with TSHR-adenovirus (present study) or vaccinated with TSHR-DNA [10] are located in the TSHR A-subunit (Fig. 5). We anticipated T cell epitope spreading because of the enhanced serum antibody levels after three versus one TSHR-adenovirus injections. Likewise, we expected to find an expanded T cell epitopic repertoire in antibody positive TSHR-adenovirus immunized mice compared with ‘naked’ TSHR-DNA vaccinated BALB/c mice, the majority of which were antibody negative [10]. However, despite enhanced responses to multiple peptides after the third injection, particularly in hyperthyroid mice, the pattern was more consistent with increased background ‘noise’ than with spreading from primary epitopes to dominant secondary epitopes. It is possible that the epitopes recognized by T cells that provide help to B cells producing functional antibodies (TSAb) vary from mouse to mouse. Alternatively, they may provide signals that are too low for detection in our assay. Overall, however, the similarity between responses from animals immunized in different ways (plasmid versus adenovirus vectors, one versus three immunizations) demonstrates the power of the MHC in determining T cell epitopes recognized by inbred mouse strains.

Fig. 5.

Fig. 5

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Amino-acid sequences and location on the TSHR ectodomain of the T and B cell epitopes recognized by TSHR-adenovirus immunized BALB/c mice. Shaded boxes indicate the three T cell epitopes. Amino acids comprising the A-subunit, or the ectodomain portion of the B-subunit, are boxed. Linear antibody epitopes [21] are in italics and underlined; the dominant epitope is at the amino-terminus and lesser epitopes towards the C-terminus of the ectodomain. Because signal peptide residues 1–21 are cleaved from the mature protein, the first synthetic TSHR peptides (peptide A) spans residues 22–41. The TSHR amino acid sequence is from reference [29].

There were some notable differences between the cytokine responses of BALB/c mice immunized with TSHR-adenovirus (present study) versus our previous observations for TSHR-DNA [7,9,10]. First, maximal IFN-γ responses to challenge with TSHR-antigen were higher in splenocytes from mice immunized three times with TSHR-adenovirus than with TSHR-DNA (10 000 versus∼3000 pg/ml). Second, TSHR-antigen induced low levels of IL-2 by splenocytes from TSHR-DNA-vaccinated mice [9,10] but not from TSHR-adenovirus immunized mice. It is conceivable that, in the powerful recall response of adenovirus-mice, IL-2 produced at an early stage was depleted at the time point used to measure IFN-γ production. Third, secretion of TNF-α in response to TSHR-antigen was observed in TSHR-DNA but not control mice [10]. The potency of the adenovirus vector to stimulate TNF-α secretion by macrophages and dendritic cells [19] may account for constitutive production of this cytokine by splenocytes from all mice injected with adenovirus. Moreover, dendritic cell maturation induced by adenovirus [20] may explain the ‘primary’in vitro response (TNF-α) by splenocytes from control-adenovirus-immunized mice to TSHR antigen. Finally, the presence of IL-5 in TSHR-stimulated cultures of splenocytes, albeit from a single TSHR-adenovirus immunized mouse, points to the involvement of Th2 cytokines, consistent with our observations for murine IgG1 subclass TSHR antibodies, as well as IL-4 mediated down-regulation of hyperthyroidism, in the adenovirus Graves’ disease model [18].

Recently, we showed that the dominant linear antibody epitope recognized by BALB/c mice immunized with TSHR-adenovirus (and TSHR-DNA) lies at the extreme amino-terminus of the TSHR ectodomain [21]. Overlapping peptides C and D (residues 52–71 and 67–86) recognized by T cells are close to, but do not include, this B cell epitope (Fig. 5). The third peptide recognized by T cells (peptide J, residues 157–176) lies within the leucine rich repeat region of the ectodomain. Overall, there is no overlap between T cell epitopes and the dominant linear B cell epitope recognized by TSHR-adenovirus immunized mice, although all are located in the TSHR A-subunit. Unlike the lack of T cell epitope spreading to other dominant region(s), three TSHR-adenovirus injections led to linear antibody epitope spreading from the amino terminus to several minor epitopes (spanning residues 352–401) that encompass the C-peptide region (excised from the two subunit form of the receptor) and part of the B subunit. Regarding conformational epitopes, studies using chimeric TSH/LHCG receptors have narrowed recognition by TSAb in mice immunized with fibroblasts coexpressing the TSHR and MHC class II to the amino terminal half of the TSHR ectodomain [22,23], as has also been shown for human autoantibodies (for example [24–27]. However, the precise location of conformational TSAb epitopes and TSAb-specific T cell epitopes remain unknown.

The restricted number of T cell epitopes and lack of spreading, at least to dominant epitopes, pose the following question: How can we account for the remarkable efficacy of TSHR-adenovirus, compared with TSHR-plasmid DNA, to induce TSAb and hyperthyroidism in the same mouse strain? We suggest that the answer lies not in T cell epitope specificity but rather in the quantity and/or quality (conformation) of the TSHR antigen available for B cell recognition. In terms of the level of TSHR expression, intramuscular injection of TSHR-adenovirus, but not TSHR-plasmid DNA, induces sufficient TSHR for detection of radiolabelled TSH binding [1]. Such TSH binding also indicates that the expressed receptors are conformationally intact. It is well recognized that TSHR conformation is critical for TSH and TSAb recognition in humans (reviewed in [28]. B cells capable of producing such antibodies will need to be driven by TSHR antigen in the correct conformation. The quality of the lesser amount of TSHR generated by intramuscular naked DNA vaccination is unknown. TSHR-DNA vaccination is usually performed after pretreatment with cardiotoxin or coinjection with sucrose, approaches used to induce muscle damage and enhance uptake by appropriate antigen presenting cells. Such conditions may alter TSHR conformation and affect TSAb production but would have no effect on the presentation of linear peptides to T cells.

In conclusion, contrary to expectation, we found no evidence of spreading from primary dominant to secondary dominant T cell epitopes in mice immunized with TSHR-adenovirus. Moreover, the same primary immunodominant epitopes were recognized by hyperthyroid and by euthyroid mice. These data indicate that factors other than particular T cell epitopes play a role in generating TSAb with consequent hyperthyroidism. We suggest that a contributing factor to the efficacy of TSHR-adenovirus immunization may be the expression of significant quantities of conformationally intact TSHR protein suitable for driving TSAb-specific B cells.

Acknowledgments

This work was supported by National Institutes of Health Grants DK 54684 (S.M.M) and DK 19289 (B.R). We thank Dr Boris Catz (Los Angeles, CA) for his generous support and Dr John C. Morris III, Division of Endocrinology, Mayo Clinic, Rochester MN, for providing us with his panel of TSHR peptides.

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