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The Journal of Immunology, 2002, 169: 5332-5337.
Copyright © 2002 by The American Association of Immunologists

Delineation of Five Thyroglobulin T Cell Epitopes with Pathogenic Potential in Experimental Autoimmune Thyroiditis1

Panayotis Verginis, Marianne M. Stanford and George Carayanniotis2

Division of Endocrinology, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Experimental autoimmune thyroiditis (EAT) is a T cell-mediated disease that can be induced in mice after challenge with thyroglobulin (Tg) or Tg peptides. To date, five pathogenic Tg peptides have been identified, four of which are clustered toward the C-terminal end. Because susceptibility to EAT is under control of H-2Ak genes, we have used an algorithm-based approach to identify Ak-binding peptides with pathogenic potential within mouse Tg. Eight candidate synthetic peptides, varying in size from 9 to 15 aa, were tested and five of those (p306, p1579, p1826, p2102, and p2596) were found to induce EAT in CBA/J (H-2k) mice either after direct challenge with peptide in adjuvant or by adoptive transfer of peptide-sensitized lymph node cells (LNCs) into naive hosts. These pathogenic peptides were immunogenic at the T cell level, eliciting specific LNC proliferative responses and IL-2 and/or IFN-{gamma} secretion in recall assays in vitro, but contained nondominant epitopes. All immunogenic peptides were confirmed as Ak binders because peptide-specific LNC proliferation was blocked by an Ak-specific mAb, but not by a control mAb. Peptide-specific serum IgG was induced only by p2102 and p2596, but these Abs did not bind to intact mouse Tg. This study reaffirms the predictive value of Ak-binding motifs in epitope mapping and doubles the number of known pathogenic T cell determinants in Tg that are now found scattered throughout the length of this large autoantigen. This knowledge may contribute toward our understanding of the pathogenesis of autoimmune thyroiditis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Experimental autoimmune thyroiditis (EAT)3 can be induced in genetically susceptible mice after challenge with thyroglobulin (Tg) in adjuvant (1, 2, 3, 4). A salient feature of the disease is the gradual destruction of the thyroid gland by infiltrating mononuclear cells, a process which also takes place in Hashimoto’s thyroiditis in humans, leading to hypothyroidism (3). There is strong evidence that CD4+ T cells are both sufficient and necessary in EAT induction because the disease can be transferred into naive recipients by CD4+ T cell lines or clones (5, 6), does not develop in nude mice (7), and is under the control of H-2Ak genes in mice bearing the susceptible H-2k haplotype (8, 9, 10).

Evidence supporting the view that Tg is a major autoantigen in clinical or experimental thyroiditis dates back to 1956 (11, 12). The molecule is quite abundant in the thyroid gland, representing 75–80% of the total protein in the thyroid extract (13), and it can be easily isolated by gel filtration chromatography. These features, however, did not aid in the identification of pathogenic T cell epitope sites by biochemical methods, as this was hampered by the large size of the molecule (homodimeric mass = 660 kDa). In addition, previous T cell epitope mapping efforts could not avail themselves of the mouse Tg (mTg) gene sequence information and used either the human (14) or the partial rat (15) Tg sequence data in EAT studies, relying on the high sequence homology among Tg from different species. Despite these difficulties, over the last decade and via a variety of methods, five immunopathogenic Tg peptides have been identified, encompassing at least six distinct T cell epitopes (16).

The use of Tg peptides as model Ags in EAT generated an impetus for studying the immunoregulation of the disease (17). At the same time, the emerged map of the pathogenic Tg epitopes focused attention on new issues that remain unresolved. First, none of the known pathogenic Tg peptides appear to comprise an immunodominant epitope, because these peptides cannot be generated after processing of intact Tg by APCs in vitro (16, 17). The experimental evidence, however, clearly suggests the presence of dominant Ak-binding epitopes(s) within Tg, as supported by the known genetic control of the Tg-mediated EAT by the I-Ak locus (8, 10) and the prevention of EAT by treatment of mice with Ak-specific Abs (18). Second, four of the five known pathogenic peptides are clustered toward the C-terminal end of Tg, raising the question of whether the rest of this large molecule can contain additional EAT-causing epitopes.

In this study, we proceeded to do a systematic search of the complete mTg sequence (19) for the detection of dominant and/or additional EAT-causing T cell epitopes by using an algorithm (20) that searches for Ak-binding motifs within a protein sequence. This algorithm takes into account the physicochemical characteristics and structural properties of amino acids within motifs that are shared among immunogenic Ak-binding peptides. The study was undertaken in the EAT-susceptible strain CBA/J (H-2k), but suggests an approach that can be applied equally well to EAT-susceptible strains of other H-2 haplotypes or to other thyroid autoantigens.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and Ags

Female CBA/J (H-2k) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were used in experiments at 6–8 wk of age. Tg was extracted from thyroids of outbred ICR mice as previously described (21). Briefly, frozen glands (Bioproducts for Science, Indianapolis, IN) were homogenized in phosphate buffer, pH 7.0, and the supernatant was centrifuged three times at 16,000 x g. Tg was obtained from the supernatant by gel filtration using Sepharose CL-4B (Pharmacia, Baie d’Urfé, Quebec, Canada). The fractions of peak II were pooled, concentrated to 3–5 mg of PBS, filter sterilized, and stored at -20°C until use. Tg concentrations are expressed as the molarity of the monomeric form (330 kDa). The Tg peptide (2495–2511) GLINRAKAVKQFEESQG (p2495) was synthesized at the Alberta Peptide Institute (Edmonton, Alberta, Canada), whereas all other Tg peptides were synthesized by Sigma-Genosys (The Woodlands, TX). All peptides were blocked with an acetyl group at the N-terminal and with an amide group at the C-terminal, whereas the thiol group of internal Cys residues was blocked by acetamide. All peptides were used in experiments at >80% purity.

T cell activation assays

Mice were immunized s.c. under ether anesthesia with 100 nmol of peptide or 100 µg of Tg in 100 µl of 1:1 PBS/CFA (with Mycobacterium butyricum; Difco Laboratories, Detroit, MI) emulsion. Nine days later, the inguinal, brachial, and axillary lymph nodes were collected aseptically and single cell suspensions were prepared in DMEM supplemented with 10% FBS (Cansera, Ontario, CA), 20 mM HEPES buffer, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Life Technologies, Rockville, MD), and 5 x 10-5 M 2-ME (Sigma-Aldrich, St. Louis, MO). After centrifugation and washing, cells (4 x 105 cells/200 µl/well) were cultured in the presence or absence of Ag in flat-bottom 96-well plates and incubated for 4 days at 37°C in a 10% CO2, 90% air-humidified incubator. Eighteen hours before harvesting, 1 µCi of [3H]thymidine (6.7 Ci/mmol; DuPont, Mississauga, ON, Canada) was added to each well in 25 µl of complete medium. The cells were harvested using a Harvester 96 Mach III M (Tomtec, Hamden, CT), and incorporated radioactivity was measured using the TopCount NXT microplate counter (Canberra Packard Canada, Mississauga, ON, Canada). Stimulation index (S.I.) is defined as follows: (cpm in the presence of peptide)/(cpm in the absence of peptide). mAbs were purified by affinity chromatography on protein G-Sepharose 4 Fast Flow columns (Pharmacia) from culture supernatants of the hybridomas 10-3.6.2 (IgG2a) reactive with I-Ak (22) and H16-L10-4R5 (IgG2a) specific for the influenza A nucleoprotein (23) purchased from the American Type Culture Collection (Manassas, VA). Inhibition of proliferation was performed at 10 µg/ml final concentration of the blocking mAb, a dose that has been previously shown to be nontoxic and effective in the blockade of T cell hybridoma clones (24). The data are expressed as follows: % inhibition = [1 - (cpm in the presence of mAb)/(cpm in the absence of mAb)] x 100.

Detection of cytokines and peptide-specific IgG by ELISA

Cytokine production was determined in culture supernatants harvested after 48-h stimulation of lymph node cells (LNCs) with Ag (8–20 µM). Detection of IL-2, IL-4, IL-10, and IFN-{gamma} was performed by sandwich ELISA based on noncompeting pairs of capture and detection (biotinylated) mAbs as follows: IL-2, JES6-1A12, and JES6-5H4; IFN-{gamma}, R4-6A2, and XMG1.2 (BD PharMingen, San Diego, CA); IL-4, 11B11 (American Type Culture Collection), and DVD-6-24G2 (BD PharMingen). IL-10 was detected via the use of affinity-purified polyclonal rabbit Ab 500-P60 and 500-P60Bt (PeproTech, Rocky Hill, NJ). Alkaline phosphatase-conjugated streptavidin was purchased from Sigma-Aldrich. Standard curves were generated for each individual cytokine using known amounts of murine rIL-2 and rIFN-{gamma} (BD PharMingen) or rIL-4 and rIL-10 (PeproTech). The detection limits were 4 pg/ml for IL-2 and IFN-{gamma}, 10 pg/ml for IL-4, and 17 pg/ml for IL-10. The presence of peptide-specific IgG in pooled sera was determined by ELISA as previously described (21), using an alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma-Aldrich) as the second Ab. Light absorption of the p-nitrophenolate product at 405 nm was measured using a Vmax plate reader (Molecular Devices, Sunnyvale, CA).

Induction and histological assessment of EAT

Mice were challenged s.c. with 100 nmol of each peptide in CFA emulsion and were boosted 21 days later with 50 nmol of peptide in IFA. EAT was assessed 35 days after the initial challenge. Adoptive transfer of thyroiditis was performed as previously described (25). Briefly, LNCs from peptide-primed donor mice were cultured for 72 h in the presence of 20 µM of the respective peptide. The cells were then harvested, and after washing three times, 2 x 107 cells in 200 µl of PBS were injected i.p. into syngeneic recipients (six mice per group). EAT was assessed 14 days post-transfer. Fixation, embedding, and sectioning of thyroids were performed as previously described (25). Histological sections were stained with H&E, and the mononuclear cell infiltration index (I.I.) was scored as follows: 0, no infiltration; 1, interstitial accumulation of cells between two or three follicles; 2, one or two foci of cells at least the size of one follicle; 3, extensive infiltration 10–40% of total area; 4, extensive infiltration 40–80% of total area; and 5, extensive infiltration >80% of total area.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Prediction of Tg peptides containing I-Ak-binding motifs

The complete mTg sequence (19) was scanned for the presence of two Ak-binding motifs, a heptamer motif A and a pentamer motif B (Table IGo), by using the algorithm described by Altuvia et al. (20). This computerized method was developed following a compilation of an extended database of helper T cell sites and takes into account physical-chemical and structural properties of peptides (dictated by the primary amino acid sequence) that may be responsible for binding to MHC class II Ags. Sixty-nine and 47 sites containing motif A or B, respectively, were identified (data not shown). To maximize the chances for success, we focused our attention to thirteen peptides encompassing completely overlapping motifs A and B flanked by 4 aa (Table IGo). Of these, peptides (224–238) and (228–242) identified an overlapping motif-rich site, thus prompting the synthesis of a single peptide (226–239) containing this region. The peptides (824–838) with three proline residues flanking the motifs, and (837–851) with a proline residue inside a motif, were not considered for further study because of concerns that Pro may drastically affect the secondary structure. Peptides (2490–2504) and (2543–2557) were shown previously to contain EAT-causing T cell epitopes (21, 26, 33). With these considerations in mind, we proceeded to synthesize the eight motif A and B-containing peptides (p110, p226, p306, p1579, p1826, p2026, p2102, and p2596; Table IGo) and examine their immunopathogenic properties.


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  		Table I. Putative Ak-binding peptides within the mTg sequence

 
Identification of T cell-activating peptides encompassing Ak-binding epitopes

To examine the immunogenicity of the Tg peptides, CBA/J mice were challenged s.c. at two sites on the back with 100 nmol of each peptide. Nine days later, the inguinal, axillary, and brachial LNCs were collected and cultured in the presence of varying concentrations of the immunizing peptide. Five of eight Tg peptides, p306, p1579, p1826, p2102, and p2596 (Fig. 1GoA), induced significant and specific LNC proliferation because there was no detectable response against the control peptide p2495. This reactivity profile correlated well with the capacity of the same peptides to elicit IL-2 (Fig. 1GoB) and/or IFN-{gamma} (Fig. 1GoC) release from such activated LNCs. IL-4 and IL-10 were undetectable in all culture supernatants (data not shown), indicating that these peptides—with the exception of p2102, which elicited IL-2 but not IFN-{gamma} release—activated Th1 cells. In all cultures, peptide-specific proliferation was significantly blocked by an I-Ak-specific mAb, but not by a control nucleoprotein-specific mAb (Fig. 2Go), strongly suggesting that recognition of these five immunogenic peptides occurred in the context of Ak molecules. These results confirmed the predictive value of the algorithm and the identification of five new Tg peptides encompassing Ak-restricted T cell epitopes.



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  		FIGURE 1. Assessment of peptide immunogenicity in CBA/J mice. A, Recall proliferative responses of peptide-primed LNCs in the presence of 25 µM of the respective peptide or p2495 as a control. Data represent the mean S.I. values of triplicate wells that were obtained from full peptide titration curves and are representative of four experiments. Background cpm ranged between 500 and 2000. B and C, Determination of cytokines by sandwich ELISA in 48-h culture supernatants of peptide-primed LNCs incubated in the presence of 10–20 µM of the respective peptide or p2495 (control). IL-4 and IL-10 were undetectable (with detection limits of 10 and 17 pg/ml, respectively) under similar conditions. Results are representative of two separate experiments.

 


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  		FIGURE 2. Inhibition of LNC proliferative responses against various Tg peptides (final concentration, 25 µM) in the presence of mAbs (10 µg/ml) specific for I-Ak or influenza nucleoprotein (control). Data represent mean ± SD of triplicate wells.

 
Immunogenic Tg peptides do not contain immunodominant determinants

To examine whether any of the five immunogenic peptides contain dominant T cell determinants, CBA/J mice were s.c. primed with 100 µg of intact Tg in CFA as above, and 9 days later the draining LNCs were cultured in the presence of Tg or free peptide. As shown in Fig. 3Go, Tg-primed LNCs responded strongly to Tg in vitro but failed to respond to equimolar (0.2–0.9 µM range) concentrations of free peptide. This lack of responsiveness was observed even at higher concentrations of free peptide (up to 75 µM), thus excluding the possibility that a narrow range of peptide concentration might influence the results. Conversely, LNCs primed in vivo with any of the five immunogenic peptides did not respond to intact Tg in recall assays in vitro (data not shown). These results do not support the view that these immunogenic peptides encompass dominant T cell epitopes.



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  		FIGURE 3. Proliferative reponses of mTg-primed LNCs to the indicated Ags in vitro. Data show the mean ± SD of S.I. values of triplicate wells and are representative of two experiments. Background cpm was 2000.

 
The I-Ak-binding peptides are thyroiditogenic in CBA/J mice

Subsequently, CBA/J mice (six per group) were immunized with each of the five immunogenic peptides above in CFA and were boosted, 3 wk later, with the same peptide in IFA. Five weeks from the initial challenge, thyroid glands were removed for histological examination of EAT development, and the I.I. was scored as described in Materials and Methods and representatively shown in Fig. 4Go. With the exception of p2102, which did not elicit detectable pathology, all other peptides induced mild and variable EAT, with mean I.I. varying from 0.2 (p1826) to 1.2 (p306) (Table IIGo). In contrast, adoptive transfer of peptide-primed LNCs into syngeneic CBA/J hosts increased both the incidence and the severity (I.I. 0.7–2.2) of EAT in all groups, including the one challenged with p2102 (Table IIGo), confirming that all five immunogenic peptides have thyroiditogenic potential. Intrathyroidal homing of mononuclear cells was specific because analogous infiltration was not observed in liver or kidney samples of these mice (data not shown). Peptide pathogenicity did not correlate with the presence of peptide-specific IgG in pooled sera of CBA/J mice with EAT, because only p2102 and p2596 elicited IgG responses (Fig. 5Go). In addition, peptide-reactive IgG did not appear to bind to intact Tg (Fig. 5Go), suggesting either that p2102 and p2596 are not expressed on the surface of Tg or that they adopt in free form a conformation different from the one they assume within the intact Tg molecule.



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  		FIGURE 4. Histological appearance of mononuclear cell infiltration in mouse thyroids after induction of EAT with Tg peptides. A, Normal gland; I.I. = 0. B and C, Interstitial accumulation of inflammatory cells; I.I. = 1. D and E, One or two foci of inflammatory cells; I.I. = 2. F, Extensive infiltration, 10–40% of total thyroid gland; I.I. = 3. Magnifications: A, B, D, and F, x100; C and E, x200.

 

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  		Table II. EAT induction by Tg peptides in CBA/J mice

 


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  		FIGURE 5. IgG responses in pooled immune sera of CBA/J mice (six mice/group) that were primed and boosted with the indicated peptide for EAT development as described in Table IIGo. Mice were bled 5 wk after the initial challenge. The reactivities against the immunizing peptide, mTg, or p2495 (control) are presented as the mean OD values of triplicate wells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have conducted an algorithm-based search for pathogenic, I-Ak -binding peptides within the complete mTg sequence to substantiate, at the T cell epitope level, the well-known control of Tg-induced mouse EAT by the I-Ak region (8, 10). Our findings double the known number of EAT-inducing Tg sequences from five to 10 and map, for the first time, four MHC class II-binding pathogenic peptides outside the 534-aa C-terminal acetylcholinesterase-homologous region of Tg (27). These four peptides (p306, p1579, p1826, and p2102) are scattered throughout the 2200-aa N-terminal segment of the Tg molecule (Fig. 6Go), which is known to contain three families of homologous domains with cysteine-rich motifs (14, 28). Interestingly, p306 is localized within the I.4 domain with type 1 homology, whereas p1579 as well as p2102 are encompassed, respectively, in the IIIa.1 and IIIa.3 domains that express type 3 homology (Table IIIGo). The fifth pathogenic peptide, p2596, maps in the vicinity of the (2549–2560) epitope, which encompasses a thyroxine molecule at position 2553 (26).



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  		FIGURE 6. Relative positions of Tg peptides that are known to cause EAT. , Peptides identified in the present report; numbers denote amino acid coordinates within the mTg molecule (19 ) without including the 20 aa leader sequence. {blacksquare}, no asterisk: Peptides identified in previous studies with their amino acid coordinates as originally published and in agreement with the mTg sequence according to Ref. 31 . {blacksquare} with asterisk: Peptides from the human Tg sequence (14, 16).

 

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  		Table III. Positioning of pathogenic peptides within the repetitive domains of mouse Tg

 
The reasons for the lack of immunogenicity of p110, p226, and p2026 are not known but may relate to non-H-2 effects on immune responsiveness or influences of motif-flanking residues on T cell recognition (29). In competitive inhibition experiments using a cloned Ak-restricted T cell hybridoma specific for the previously identified Tg (2499–2507) epitope (24), we have not seen large differences in Ak-binding capacity among all peptides tested (data not shown). Therefore, collectively, the results confirm the predictive value of the algorithm by Altuvia et al. (20) in the identification of Ak-binding peptides. This method relies on physical-chemical and structural properties of peptides, such as hydrophobicity, charge, hydrogen bonding capability, etc., which can be extracted from amino acid sequence data. Although in this study we concentrated on candidate peptides with completely overlapping A and B motifs, it is noteworthy that 99 additional Tg sites with nonoverlapping A or B motifs were also revealed by this search (data not shown). In contrast, scanning of the complete mTg sequence with a more restrictive nonamer Ak-binding motif derived from crystallographic data of the Ak molecule complexed with a hen egg lysozyme peptide (30) identified only three sequences: (1576–1584) and (1585–1593) partially overlapping with p1579 and (2107–2115) partially overlapping with p2102 (Table IGo). Thus, it is difficult to predict from such data how many additional Ak-binding peptides are harbored within Tg, and it is likely that alternative approaches might still identify other EAT-causing determinants. Interestingly, none of the five pathogenic Tg peptides described herein overlap with seven putative I-Ek-binding peptides uniquely expressed in murine Tg, as identified by the EpiMer algorithm (31). In contrast, p306 and p2102 are being indicated as sites that contain one of five Ek-binding motifs according to the algorithm of Altuvia et al. (20).

The current data also bring into focus the fact that none of the seven known Ak-restricted pathogenic Tg peptides—five from the present study, plus the (2499–2507) and (2549–2560) epitopes (24, 26)—can be classified as immunodominant because they cannot be generated after processing of intact mTg in vivo and/or in vitro. This is a rather paradoxical finding in view of the fact that Tg-induced EAT is under control of the I-Ak locus (8, 10), implying the presence of dominant Ak-restricted T cell epitopes in this large autoantigen. The following explanations can be proposed to account for this apparent discrepancy. First, one could maintain that immunodominant T cell epitopes in Tg exist but remain unidentified because 1) the sheer size of Tg prevents their easy detection, 2) such epitopes do not contain hormonogenic sites but are normally iodinated, and 3) detection is precluded by limitations of algorithm-based approaches in epitope mapping (here and in Refs. 21 and 32) or inherent restrictions in using cloned Tg-reactive T cell hybridomas for screening the antigenicity of overlapping Tg peptides, a method followed by Champion et al. (26) for the discovery of the pathogenic (2549–2560) sequence. Second, the theoretical possibility exists that all Ak-restricted Tg peptides interact with MHC with similar affinity and activate T cell precursors of low frequency, thus not allowing a clear-cut hierarchy of immunodominance to emerge. In this case, Ak-controlled susceptibility to EAT or reactivity to mTg in vitro would be detected as a result of additive or synergistic effects of Tg peptide-specific T cell clones, which nevertheless would remain individually undetectable due to their low frequency. Third, and most likely, the conventional criteria for the definition of immunodominance might not apply to Tg. This is an explanation we have previously elaborated on (16), as it is conceivable that in vitro processing of this large autoantigen does not normally generate enough of any given epitope to activate peptide-specific lymphoid cells. For example, optimal processing of 100 µg/ml (150 nM) of intact dimeric Tg would not generate >0.6 µg/ml (300 nM) of any given 2-kDa peptide.

For most Tg peptides tested in this study, the adoptive transfer protocol led to EAT with higher incidence and severity than that induced after direct challenge of hosts with peptide in CFA. These data might be explained on the basis of a peptide dose constraint on direct EAT induction (100 nmol of peptide may be a relatively low dose given the nondominant nature of these Ags), vis-à-vis the capacity of preformed peptide-specific effector T cells to home selectively to the thyroid after the adoptive transfer protocol. In analogy with the peptide (2549–2560) previously identified by Roitt and colleagues (33), the pathogenicity of p2102 was shown only by adoptive transfer of p2102-primed LNCs to syngeneic naive CBA mice, confirming the view (16) that the thyroiditogenicity of candidate peptides should not be examined only by direct challenge of host mice with the respective epitope. We cannot explain why peptide p2102 does not elicit detectable levels of IFN-{gamma} in culture. However, its potential to elicit mild EAT via the adoptive transfer protocol indicates that p2102 activates a very low number of Th1 cells and/or other types of effector cells that can infiltrate the thyroid. Indeed, previous studies have shown that mice lacking the IFN-{gamma} receptor gene are able to develop disease upon challenge with Tg (34), and adoptive transfer of Tg-activated T-cells along with an IFN-{gamma}-specific Ab into naive hosts results in the induction of granulomatous EAT (35).

The present findings support the notion that Tg encompasses many noniodinated T cell epitopes, which can be cryptic but pathogenic under conditions that allow their generation and presentation in professional APCs. In that regard, we have previously shown that processing of highly iodinated Tg (36) or Tg-Ab complexes (37) in APCs allows selective presentation of pathogenic but cryptic Tg peptides. These mechanisms would promote epitope spreading (38, 39) and rapid emergence of thyroiditogenic T cells because it is unlikely that peripheral tolerance would have been pre-established against cryptic Tg determinants (39). Some epitopes may even promote expansion of autoreactive T cells via molecular mimicry with foreign pathogens, as has been shown previously with the pathogenic Tg (2695–2706) determinant that exhibits high homology with the (368–381) peptide of murine adenovirus type 1 sequence (25). In contrast, an immunoregulatory role for peptide-reactive IgG Abs is quite unlikely for the Tg sequences described herein, because only p2102 and p2596 elicited IgG responses, and these Abs did not bind to intact Tg.

In conclusion, the present study has established the immunopathogenic potential of five novel Tg peptides in CBA/J mice out of a list of eight candidate sequences that contain Ak-binding motifs. The overall importance of all sequences in mouse EAT can be further evaluated in future studies using H-2k strains of diverse non-H-2 backgrounds and/or strains of other H-2 haplotypes. Work with previously identified pathogenic Tg epitopes, such as the 9-mer (2496–2504), which binds to nonisotypic I-Ek and I-As determinants (24), and the 11-mer (2549–2559), which induces EAT in CBA/J but not in DBA/1 (H-2q) or SJL (H-2s) mice (40), suggests that the outcome of such studies is impossible to predict. However, our findings demonstrate a promising approach for the identification of immunopathogenic Tg peptides with MHC class II-binding properties in EAT that can be analogously applied in the search of Tg epitopes relevant to human thyroiditis.


   Acknowledgments
 
We thank Judy Foote and Howard Gladney for their expert help with the histology work.


   Footnotes
 
1 This work was supported by a grant from the Canadian Institutes of Health Research. P.V. and M.M.S. were supported by stipends from the Canadian Institutes of Health Research and the Memorial University of Newfoundland. Back

2 Address correspondence and reprint requests to Dr. George Carayanniotis, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada, A1B 3V6. E-mail address: gcarayan{at}mun.ca Back

3 Abbreviations used in this paper: EAT, experimental autoimmune thyroiditis; Tg, thyroglobulin; mTg, mouse Tg; S.I., stimulation index; LNC, lymph node cell; I.I., infiltration index. Back

Received for publication June 19, 2002. Accepted for publication August 30, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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