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Endocrinology Vol. 147, No. 6 3085-3092
Copyright © 2006 by The Endocrine Society

Induction of Autoimmune Thyroiditis and Hypothyroidism by Immunization of Immunoactive T Cell Epitope of Thyroid Peroxidase

H. P. Ng and Annie W. C. Kung

Department of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China

Address all correspondence and requests for reprints to: Annie W.C. Kung, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam, Hong Kong, China. E-mail: awckung{at}hkucc.hku.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Autoimmune thyroiditis (AT) is characterized by a continuous inflammatory self-destructive process that eventually leads to chronic progressive dysfunction of the thyroid. In a previously established experimental AT model, C57bl/6 mice immunized with recombinant mouse thyroid peroxidase (TPO) (rmTPO) developed lymphocytic thyroiditis and anti-TPO antibody but not chronic hypothyroidism. To determine the immunodominant epitope(s) of TPO, T cell proliferation assays were performed in which rmTPO-primed lymph nodes cells were reacted with recombinant mTPO fragments or short overlapping synthetic TPO peptides. Within residue 405–849, peptide 540–559 gave the maximum proliferation response with a stimulation index more than 12. Mice immunized with peptide 540–559 developed antibody against rmTPO and native mouse TPO protein, lymphocytic thyroiditis, and hypothyroidism. In conclusion, this study demonstrated that TPO is the autoantigen for the development of lymphocyte thyroiditis and thyroid dysfunction, and peptide 540–559 is the immunodominant T cell epitope of TPO. Identification of T cell epitopes of TPO may enable the development of immunotherapy to prevent chronic hypothyroidism in AT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AUTOIMMUNE DISORDERS ARE common human diseases with autoimmune thyroid diseases (AITDs), including Hashimoto’s thyroiditis (HT) and Graves’ disease (GD), affecting 2–3% of the population, especially females (1). HT is characterized by a continuous inflammatory self-destructive process that eventually leads to chronic hypothyroidism (2). Thyroid peroxidase (TPO) expressed on the thyroid cell surface is a major thyroid autoantigen (3, 4, 5), and both humoral and cell-mediated immune responses against TPO are believed to be responsible for the pathogenesis of HT (6, 7, 8).

Patients with HT have high titers of anti-TPO autoantibodies. TPO autoantibodies can activate complement cascade (9, 10) and can mediate the antibody-dependent cell cytotoxic on thyrocytes in vitro (11, 12, 13, 14). There is interesting evidence showing that TPO-specific B cells are able to act as antigen-presenting cells. TPO autoantibodies with different epitopes can direct different antigenic determinants to T cells (15, 16, 17). Importantly, TPO autoantibodies from patients with AITD are found to have a remarkable feature of being restricted to epitopes within immunodominant regions A and B (18, 19, 20), implying that a set of unique disease-related autoantibodies may exist for AITD development.

TPO autoreactive T cells are tightly regulated by several mechanisms of tolerance. The high-affinity T cell clones against TPO are normally deleted during development. Although TPO-specific T cells with low-affinity T cell receptor (TCR) can escape deletion and keep circulating in the body, their activation are restricted in a normal physiological condition. Under this circumstance, TPO autoantibodies can help present TPO efficiently even if only a minute amount of TPO is released during normal turnover of thyrocytes or local inflammation. TPO autoreactive T cells also play a key role in the pathogenesis of HT. Besides helping production of TPO antibodies (21, 22), TPO-specific T cells cause thyroid destruction through mechanisms including either direct cytotoxicity mediated by CD4+ and CD8+ T cells or programmed apoptosis mediated by Fas and TNF-{alpha} (23). In a recent study to evaluate the pathogenic role of human TPO T cells in HT, mice transgenic for the human T cell receptor V gene specific against TPO cryptic epitopes 535–541 could induce spontaneous destructive thyroiditis with histological, clinical, and hormonal features comparable with human autoimmune hypothyroidism (24). Because the transgenic mice are absence of mature B cells and antibodies, this model emphasizes a key role of pathogenic self-reactive T cells in the initiation of autoimmunity.

Identification of T cell epitopes of TPO is important in understanding the pathological role of T cells in the initiation of autoimmune thyroiditis (AT). In human AITD, TPO-specific T lymphocytes collected from various sources including the thyroid gland, lymph nodes draining the thyroid, and peripheral blood lymphocytes have been studied (25). The T cell epitopes of TPO identified from these studies varied in their location (26, 27, 28, 29, 30). It is likely that by the time patients presented clinically, the disease was at its late stage, and T cells involved in the early stage of the disease cannot be distinctly identified due to possible epitope spreading as the disease progresses. To this end, experimental AT (EAT) models are valuable tools for studying the pathogenesis of autoimmunity. In our previous studies, we were able to elicit specific T and B cell response against recombinant mouse TPO (rmTPO) in C57bl/6 mice immunized with rmTPO protein (31). In the present study, we attempted to identify the immunodominant peptide of mTPO using this mTPO-induced EAT model to gain a better understanding of the role of pathogenic T cells in AITD.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Production of rmTPO ectodomain and TPO fragment
rmTPO was produced from Escherichia coli as previously described (31). Briefly, mTPO ectodomain cDNA was introduced into the polylinker of vector pGEX-4T-1, downstream from the glutathione-S-transferase (GST) gene. Expression of a GST-mTPO fusion protein and GST were induced by 0.1 mM isopropylthiogalactoside (IPTG). Cells were harvested by centrifugation, pellets were resuspended in 20 ml PBS containing 1% Sarkosyl, 1 mM phenylmethylsulfonylfluoride (PMSF), 1% aprotinin, and 50 mg DNAse I, sonicated twice for 1 min, and GST-mTPO fusion proteins were purified with gluatathione-agaorose beads column. rmTPO was separated from the fusion protein by digestion with thrombin. Purified recombinant mTPO ectodomain was analyzed by western blotting using rabbit antihuman TPO polyclonal antibody Rb6.

Recombinant short mTPO fragments for proliferation assay were produced using four pairs of primer to amplify the following regions: for amino acids (aa) 1–223, forward primer 5'-gcggaattcatgagaacacttggagctatc-3', reverse primer 5'-gcggaattcaaaatcagagtactggtcatc-3'; aa 219–447, forward 5'-gcggaattctactctgattttctgccggtg-3', reverse 5'-gcggaattaggacccaggattttggggat-3'; aa 446–544, forward 5'-gcggaattcggtcctgatgccttcaggcag-3', reverse 5'-gcggaattccatcagctgcccttggacttg-3'; and aa 544–837, forward primer 5'-gcggaattcaatgaggagctgaccgagagg-3, reverse 5'-gcggaattcggatgcccgaggtagcctgcc-3'. DNA fragments amplified from each pair of primer were digested with EcoRI, and the digested DNA fragments were ligated with EcoRI-digested pGEX-4t-1 (Amersham, Buckinghamshire, UK). The recombinant plasmids were transformed into the E. coli. Transformed E. coli was induced to produce fusion protein by adding appropriated concentration of IPTG. After IPTG induction, molecular weight of each recombinant mTPO fragment was analyzed by SDS-PAGE. In the production and purification of rmTPO fragments, expression of a GST-mTPO fragments were induced by 0.1 mM (IPTG). Cells were harvested by centrifugation, and pellets were resuspended in 20 ml PBS containing 1% Sarkosyl, 1 mM PMSF, 1% aprotinin, and 50 mg DNAse I, sonicated twice for 1 min. Fusion proteins were purified with gluatathione-agaorose beads, and purified rmTPO fragments were eluted after thrombin digestions. Eluted rmTPO fragments were dialyzed three times against PBS before use in the assay.

The thyroid microsomes were prepared as described previously (32). Briefly, crude extract of native mTPO was extracted from the thyroid of 20 hypothyroid mice fed with 0.1% propylthiouracil for 10 d. Murine thyroid glands were homogenized in lysis buffer (5 M KCl containing 1 mM PMSF and 10% apotinin). The lysate was centrifuged at 100,000 x g for 1 h, and the microsomes were resuspended in lysis buffers.

Synthesis of mouse TPO peptides
Synthetic oligopeptides for in vitro T cell proliferation assays were comprised of 30 overlapping peptides, each having 20 and five aa overlapping with the adjacent peptide (Mimotopes Pty Ltd, Clayton, Victoria). The purity of these synthetic peptides was more than 70%. Peptides were numbered sequentially (1–30) beginning with aa residue 405 (Table 1Go). All peptides were dissolved in appropriate solvent (0.1% acetic acid for soluble peptide; 10–20% acetic acid for peptide with positively charge; 1–10 µM ammonium bicarbonate for peptide with negatively charge) according to the recommendation provided by the manufacturer.


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  		TABLE 1. Panel of synthetic overlapping peptides encompassing mTPO amino and residue 405–849 used for T cell proliferation study

 
Immunization of animals
Eight-week female C57b1/6 (H-2b) mice were purchased from and housed in the Laboratory Animal Unit of Faculty of Medicine (University of Hong Kong) under sterile barrier-free controlled conditions. The mTPO synthetic peptides for immunization were purchased from Mimotopes Pty Ltd and dissolved in 10 µM ammonia bicarbonate. The purity of the peptides used in immunization was more than 85%.

For identification of immunodominant peptide of mTPO, 10 mice per group were used for the experiment. rmTPO ectodomain was emulsified at a ratio of 1:1 in complete Freund’s adjuvant (CFA; Sigma, St. Louis, MO). One injection of 30 µg rmTPO ectodomain in 200 µl emulsion was applied sc in the hind footpads and at the back. For determination of immunogenicity of the peptide, 100 nmol peptide was diluted to 50 µl with PBS and emulsified with 50 µl CFA. The emulsion was injected sc into the hind footpads and at the back. The booster dose was preformed 3 wk later. Control mice were injected with peptide solvent plus CFA. Sera were collected at d 50 and 90 for antibody activity and at d 90 for total T4 and TSH levels. The study was approved by the Ethics Committee of the University of Hong Kong and performed according to the standards layout by the University of Hong Kong.

Assessment of T cell proliferation
Draining lymph nodes of rmTPO-immunized C57bl/6 mice were excised 10 d after immunization, and single-cell suspension was prepared. Cell number was adjusted to 1 x 107 cell/ml in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with L-glutamine, 100 U/ml antibiotic-antimycotic, and 10% FCS. Cell suspension (100 µl) was added to each well of a flat-bottom 96-well plate (Costar, Cambridge, MA). rmTPO ectodomain, mTPO fragments, and synthetic peptides were diluted with RPMI and added to the cell suspension at different concentrations. All experiments were performed twice, each in triplicate. After 4 d of incubation in humidified air containing 5% CO2, each well was pulsed with [methyl-3H] thymidine (Amersham). The cultured cells were then harvested on glass fiber filters, and thymidine incorporation was measured by a liquid scintillation counter. T cell proliferation response to TPO or TPO peptides was measured by 3H-thymidine incorporation and reported as counts per minute. The stimulation index (SI) was defined as:

Formula

Measurement of TPO antibody
For ELISA, either rmTPO or crude extract of native mTPO was diluted in 0.06 M sodium bicarbonate coating buffer (pH 9.6) at 10 µg/ml. As a control, 3% BSA was used for coating. Each diluted antigen (100 µl) was added to the wells of a microwell plate (Costar) and incubated overnight at 4°C. After washing three times with PBS containing 0.1% Tween 20, all wells were saturated with blocking solution (5% skimmed milk powder in PBS). One hundred microliters of 20 times diluted sera from immunized mice was added and incubated for 1.5 h at 37 C. The plate was again washed and incubated with alkaline phosphatase-conjugated antimouse IgG (Sigma) for 1 h at 37 C. The plate was washed and developed with p-nitrophenol phosphate (Sigma Fast p-nitrophenol phosphate tablet set) for 30 min at room temperature. All experiments were done in duplicate and performed twice. The absorbance was measured at 405 nm with the control wells read as blank.

Thyroid histopathology
Mice were killed at d 90 for evidence of thyroiditis. The thyroid glands were removed and carefully embedded in ornithine carbamyl transferase compound (Sakura Finetechnical Co., Tokyo, Japan), rapidly frozen in isopentane, and cooled in liquid nitrogen to generate cryostat sections. The frozen section was subjected to hematoxylin and eosin staining. The severity of thyroiditis was graded on a scale of 0–4 as follows: grade 0, normal histology; grade 1, interstitial accumulation of inflammatory cells distributed around one or two follicles; grade 2, one or more foci of inflammatory cells reaching at least the size of one follicle; grade 3, 10–40% of thyroid replaced by inflammatory cells; and grade 4, more than 40% of thyroid replaced by inflammatory cell. Scoring was performed blind to the animal treatment groups.

Serum T4 and TSH level
Serum total T4 was measured by fluorescent polarization immunoassay (Abbott Laboratories, Abbott Park, IL), and serum TSH was measured by a two-site immunometric assay (Centaur, Bayer Corp.-Diagnostics Division, Tarrytown, NY). The mean T4 and TSH levels were determined in 10 8-wk-old adult female C57bl/6 mice. The normal range was defined as 2 SD values from the mean. Mice having subnormal T4 levels or elevated TSH levels were considered as biochemically hypothyroid. The intraassay and interassay coefficients of variation for the T4 assay were 4.5 and 6.0%, respectively, and for the TSH assay were 5.5 and 6.8%, respectively.

Statistical analysis
Comparisons of thyroid function tests before and after immunization as well as comparisons between groups were by one-way ANOVA. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
T cell epitope mapping of mTPO
The mapping of T cell epitope(s) of mTPO in C57bl/6 mice was initially performed using four recombinant mTPO fragments. Lymph node cells (LNCs) draining the site of injection were obtained from C57b/6 immunized with rmTPO and stimulated with either full-length TPO or the four protein fragments. Both intact TPO and fragment D significantly stimulated LNCs to proliferate (Fig. 1Go). Fragment C also showed slight proliferation but not fragments A and B. LNCs from control mice immunized with GST protein showed no proliferative response to either full-length TPO or any of the four protein fragments. These results indicated that the major T cell epitope(s) was located within fragment D.


Figure 1
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  		FIG. 1. T cell epitope mapping of mTPO as determined by T cell proliferation assay using four recombinant mTPO fragments. rmTPO-primed LNCs from C57bl/6 mice were obtained from five mice 10 d after the second rmTPO injection. rmTPO-primed LNCs were pooled and incubated with either rmTPO ectodomain or rmTPO fragments in triplicate wells, with the experiment repeated twice. Culture condition: medium alone ({square}), 10 µg/ml rmTPO (Figure 1), 10 µg/ml mTPO fragment A (Figure 1), 10 µg/ml mTPO fragment B (Figure 1), 10 µg/ml mTPO fragment C (Figure 1), and 10 µg/ml mTPO fragment D ({blacksquare}). Results are in counts per minutes.

 
To fine map the T cell epitope(s), 30 20-mer overlapping peptides spanning mTPO aa 405–849, i.e. the region including both fragments C and D, were synthesized and reacted with LNCs primed with mTPO (Table 1Go). Among the 30 peptides, peptide p10 encoding mTPO residue 540–559 (QGQLMNEELTERLFVLSNVG) distinctly stimulated LNCs to proliferate with a SI greater than 12 (Fig. 2Go). Several other peptides including p4 (aa 480–499), p12 (aa 570–589), p13 (aa 585–604), p23 (aa 735–754), and p26 (aa 780–799) also stimulated LNCs to proliferate with an SI of around 2–2.6. Among these peptides, only p4 is within fragment C, and all the other peptides are within fragment D.


Figure 2
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  		FIG. 2. T cell epitope mapping of mTPO in C57bl/6 mice by T cell proliferation assay using 30 overlapping synthetic peptides (Table 1Go). rmTPO-primed LNCs were obtained from five C57bl/6 mice 10 d after injection of rmTPO and stimulated with the synthetic peptides at 50 nmol/ml. All experiments were preformed in triplicates and repeated twice. Results are reported in 3H thymidine incorporation as counts per minutes. The number above each histogram represents the mean SI of each peptide.

 
Antibody production against mTPO after immunization with peptide 540–559
To determinate the immunogenicity of peptide 540–559 (p10), antibody production against rmTPO was determined at d 50 and 90 after immunization. For comparison, mice were immunized with either peptide solvent (10 µM ammonia bicarbonate) alone or peptide 735–754 (p23) or peptide 795–814 (p27). The results revealed that antibody activity reacting against rmTPO was also detectable in mice immunized with peptide 735–754 but not with peptide 795–814 or solvent alone. The antibody activity with peptide 735–754 was weaker, and the response was delayed when compared with peptide 540–559 immunization.

The reactivity of the antibody against native mouse TPO protein was also determined after peptide immunization (Fig. 3BGo). Antibody activity recognizing mouse TPO was detected in mice immunized with both peptides 540–559 and 735–754, with the activity being 1.5–2.9-fold above that of d 0. None of the animals developed such antibody activity after immunization with peptide 795–814 or solvent alone.


Figure 3
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  		FIG. 3. Production of autoantibodies reacting to rmTPO (A) and mouse (B) TPO crude extract after immunization of mTPO peptides (rmTPO peptides 540–559, 735–754, and 795–814 or peptide solvent; n = 10 per group). Sera were collected at d 50 and 90 and diluted at 1:20. Diluted sera were reacted with ELISA plates coated with 100 µl of 10 µg/ml rmTPO (A) and 10 µg/ml mouse TPO (B) crude extract. Binding of antibodies was detected by antimouse IgG secondary antibody conjugated with alkaline phosphatase and visualized by developing the substrate pNpp. The OD was read at 405 nm.

 
Thyroid histology after peptide immunization
All 10 mice immunized with mTPO peptide 540–559 developed thyroiditis with mononuclear cell infiltration and destruction of thyroid follicles (Table 2Go and Fig. 4AGo). None of the control mice immunized with peptide 735–754 (Fig. 4BGo), peptide 795–814 (Fig. 4CGo), or solvent alone (Fig. 4DGo) developed histological features of thyroiditis. Immunoperoxidase staining revealed infiltration of CD3+ T cells in between thyroid follicles in peptide 540–559-immunized mice (Fig. 4EGo) but not in control mice (Fig. 4FGo).


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  		TABLE 2. Incidence of thyroiditis in C57bl/6 immunized with different TPO peptides

 

Figure 4
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  		FIG. 4. Thyroid histology of peptide-immunized C57bl/6 mice (hematoxylin and eosin stain, A–D; immunoperoxidase staining, E and F; x200). Thyroiditis with severe mononuclear cell infiltration in the thyroid was seen in mice immunized with 100 nmol rmTPO peptide 540–559 at d 90 (A). Thyroid section of control mice immunized with peptide 735–754 (B), peptide 795–814 (C), or peptide solvent (D) showing normal thyroid histology. Immunostaining showed CD3+ T cells infiltrating in between follicles in mice immunized with peptide 540–559 (E) but not in mice immunized with peptide solvent (F).

 
Serum T4 and TSH after peptide immunization
As a group, mice immunized with peptide 540–559 had significantly lower T4 levels and higher TSH levels than control mice at d 90 after immunization (both P < 0.05), with five of 10 mice having hypothyroxinemia and seven mice having elevated TSH levels (Fig. 5Go). Analysis of individual data revealed that mice having the highest thyroiditis score had the lowest serum T4 levels. None of the animals immunized with peptides 735–754 or 795–814 had abnormal T4 or TSH.


Figure 5
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  		FIG. 5. Serum total T4 (A) and TSH (B) of C57bl/6 mice at d 90 after immunization with peptides 540–559, 735–754, 795–814, or solvent. Mice immunized with mTPO peptide 540–559 had significantly lower T4 levels. Five of 10 mice had hypothyroxinemia, and seven had elevated TSH levels. The normal ranges for total T4 (55–95 nmol/liter) and TSH (0.04–0.32 mIU/liter) were established from 10 naive C57bl/6 mice of the same age.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this present study, we attempted to identify the immunodominant T cell epitopes of mTPO in a rmTPO-induced EAT mouse model, and our results showed that peptide 540–559 was able to induce thyroid autoimmunity including production of antibody against rmTPO and native mouse TPO, histological evidence of thyroiditis with mononuclear cells infiltration, and biochemical hypothyroidism.

Identification of immunodominant regions of thyroid antigens is crucial in understanding the pathogenesis of AITD. rmTPO-sensitized LNCs from C57bl/6 mice when cultured with mTPO peptides proliferated predominantly to peptide 540–559. This is a simple and direct method to map the T cell epitope(s) of the antigen, but this approach may not be suitable for all antigens, e.g. in the study of thyroglobulin (Tg), the large size of the Tg molecule (660 kDa) precludes such an approach to be adopted. Immunodominant epitopes of Tg were identified by conducting a computer-based algorithm that predicts potential mouse histocompatibility complex-binding peptides. Using such a program, Panayotis et al. (33) identified 5 Tg synthetic peptides which were able to bind H-2Ak molecules and induce EAT in susceptible CBA (H-2k) mice. However, none of the five peptides contains dominant T cell determinants because these peptides did not stimulate LNCs from Tg-primed CBA mice. In reality, natural processing of intact Tg by antigen-presenting cells does not produce these computer-predicted peptides in vivo. Other studies have adopted a similar computer-based algorithm to figure out the pathogenic human Tg peptides in humanized HLA-DR3 transgenic mouse model of AT (34). One of 39 peptides was recognized by the Tg-primed LNCs in vitro and was identified as the immunodominant peptide. Our data similarly showed that only peptide 540–559 stimulated rmTPO-primed LNCs with SI of more than 12. Interestingly, the degree of stimulation of proliferation with intact TPO was lower than that with peptide 540–559. This may be related to the lower efficiency of peptide presentation on antigen-presenting cells through processing an intact protein. Based on the significant increase in proliferation, peptide 540–559 is likely the major T cell epitope of mTPO in C57bl/6 (H2b) mice.

In the study of human TPO, Gora et al. (35) suggested that the conformational epitope is composed of at least three critical linear epitopes, i.e. P12 (aa 549–563), P14 (aa 599–617), and P18 (aa 210–225). Rabbit polyclonal antibody against these three critical linear epitopes could block the sera from patients with AITD from binding to human TPO. The percentage of inhibition from binding increased when two of the antipeptide antibodies were added together, and the greatest inhibition was seen by adding all three antipeptide antibodies into the same assay. The result of binding inhibition indicated that these three linear epitopes were located on the surface of native human TPO, and although discontinuous in aa sequence, they came very closely together when TPO was folded properly. The immunodominant peptide of mTPO residue 540–559 is likely the mouse counterpart of one of these three linear epitopes because it shares 80% homology with human epitope P12 (aa 549–563). Theoretically, mouse TPO should have a three-dimensional structure similar to that of human TPO. From this assumption, residue 540–559 of mTPO is likely located on the surface of native mTPO in vivo. Another critical linear epitope composing the conformational epitope of human TPO is P14 (aa 599–617). Its homologous peptide for mTPO is residue 587–607, sharing 72% sequence homology with its human counterpart. However, this peptide did not stimulate proliferation of mTPO-primed LNCs.

In our previous study (31), immunization of both C57bl/6 and CBA mice with rmTPO protein induced TPO autoantibody activity that binds native mouse TPO protein, although the antibody activity was higher in C57bl/6 than CBA mice. C57bl/6 was sensitive, but CBA mice were resistant to rmTPO-induced thyroiditis. Whether the development of anti-TPO autoantibodies causes susceptibility to AT has been a question of debate. The result of TPO antibody production in the peptide-immunized mice gave us some hints to address this question. In our previous study, the rmTPO antibody activity induced by immunization with rmTPO ectodomain showed no correlation with the development of thyroiditis (31). In the present study, we similarly noted that antibodies against rmTPO and native TPO protein were detectable in mice immunized with peptides 540–559 and 735–754, but only peptide 540–559 was associated with thyroiditis and hypothyroidism. Although we did not check the cytotoxicity of the TPO antibodies, the dissociation between antibody production against native TPO, i.e. pathogenic antibody, and thyroiditis suggests a lesser role of TPO autoantibodies in the initiation of thyroiditis and hypothyroidism. In comparison with rmTPO protein or Tg immunization or mice with spontaneous thyroiditis (NOD-H2h4), immunization with peptide 540–559 produced a lesser degree of thyroid follicular destruction and lower thyroiditis index.

In the determination of immunogenicity of peptide 540–559, peptide 735–754 was chosen for comparison because it produced mild stimulation of rmTPO-primed LNCs, and like peptide 540–559, it also bears two negative net charges. Peptide 540–559 induced high antibody reactivity against rmTPO, which peaked at d 50, whereas peptide 735–754 induced a slower and weaker antibody response. The different kinetics of antibody production may be related to the reactivity of peptide-specific T cells. It is likely the stronger T cell response induced by peptide 540–559 may help produce high antibody production within a shorter period. Because we did not observe the animals beyond 90 d, we are not certain whether the increasing TPO antibody activity in animals immunized with peptide 735–754 could induce thyroiditis and hypothyroidism after a longer period of observation. We believe that peptide 735–754 and other peptides that showed mild stimulation of rmTPO-primed LNCs may represent the cryptic epitopes of mTPO that stimulated T cells with low-affinity TCR, and these cryptic epitopes may also have the ability to induce thyroiditis, as described by Quarantino et al. (24). On the other hand, some cryptic epitopes may relate to activation of antigen-specific regulatory T cells, as illustrated by the inhibitory action of Tg-specific CD4+CD25+ regulatory T cells in the Tg-induced EAT model (36). We believe that the reasons for the more intense autoimmune response generated by immunization with the single immunodominant peptide 540–559 vs. the intact TPO protein is attributed to the bypass of these regulatory T cells and also to the absence of antigenic competition from other interfering peptides (36, 37).

The role of T cells in AITD is illustrated by several observations in recent publications. In a recent study of human TPO-specific T cell receptor TCR transgenic mouse model (24), the pathogenic TCR V gene specific against human TPO cryptic epitope 535–541 was introduced, and the transgenic mice spontaneously developed destructive thyroiditis with histological, clinical, and hormonal features compatible with human autoimmune hypothyroidism. Because mature B cells and antibodies were absent in this mouse model, these data emphasize a key role of pathogenic self-reactive T cells against human TPO in initiation of autoimmunity. Similarly, in the SAT transgenic mice model, spontaneous thyroiditis could be established in the Rag1+ genetic background with the presence of CD4+CD25hi regulatory T cells (37). In this TCR+Rag1+ transgenic model, TPO-specific self-reactive T cells can circumvent the function of regulatory T cells and perform their pathogenic activity. The immune environment of these TCR+Rag1+ transgenic mice mimics the human situation much more closely than that of the autoimmune TCR (Rag–/–) transgenic model, which was Rag deficient in its genetic background and lacked the function of CD4+CD25hi regulatory T cells. In our present study, immunization with immunodominant peptide 540–559 plus adjuvant is sufficient to induce both thyroiditis and hypothyroidism in wild-type C57bl/6 mice that have normal immune system, suggesting that activation of pathogenic T cell specific to a major TPO epitope and bypass of regulatory T cells are important mechanisms for the initiation of thyroid-specific autoimmunity.

In conclusion, this study demonstrated that TPO is an important thyroid antigen in initiating thyroid autoimmunity and thyroid dysfunction and that peptide 540–559 is the major T cell epitope of TPO in C57bl/6 mice. The fact that activation of a single immunodominant T cell epitope was able to induce thyroiditis and hypothyroidism emphasized the critical role of pathogenic T cells specific for TPO in the development of AT. Identification of T cell epitopes of TPO may enable the development of immunotherapy to prevent chronic hypothyroidism in patients with AITD.


   Footnotes
 
Both H. P. Ng and Annie W. C. Kung have nothing to declare.

First Published Online March 9, 2006

Abbreviations: aa, Amino acid(s); AITD, autoimmune thyroid disease; AT, autoimmune thyroiditis; CFA, complete Freund’s adjuvant; EAT, experimental AT; GST, glutathione-S-transferase; HT, Hashimoto’s thyroiditis; IPTG, isopropylthiogalactoside; LNC, lymph node cell; PMSF, phenylmethylsulfonylfluoride; rmTPO, recombinant mouse TPO; SI, stimulation index; TCR, T cell receptor; Tg, thyroglobulin; TPO, thyroid peroxidase.

Received September 2, 2005.

Accepted for publication February 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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