High salt intake does not exacerbate murine autoimmune thyroiditis.

Recent studies have shown that high salt (HS) intake exacerbates experimental autoimmune encephalomyelitis and have raised the possibility that a HS diet may comprise a risk factor for autoimmune diseases in general. In this report, we have examined whether a HS diet regimen could exacerbate murine autoimmune thyroiditis, including spontaneous autoimmune thyroiditis (SAT) in non-obese diabetic (NOD.H2(h4)) mice, experimental autoimmune thyroiditis (EAT) in C57BL/6J mice challenged with thyroglobulin (Tg) and EAT in CBA/J mice challenged with the Tg peptide (2549-2560). The physiological impact of HS intake was confirmed by enhanced water consumption and suppressed aldosterone levels in all strains. However, the HS treatment failed to significantly affect the incidence and severity of SAT or EAT or Tg-specific immunoglobulin (Ig)G levels, relative to control mice maintained on a normal salt diet. In three experimental models, these data demonstrate that HS intake does not exacerbate autoimmune thyroiditis, indicating that a HS diet is not a risk factor for all autoimmune diseases.

Maturation of dendritic cells by necrotic thyrocytes facilitates induction of experimental autoimmune thyroiditis.

Dendritic cell (DC) maturation is required for efficient presentation of autoantigens leading to autoimmunity. In this report, we have examined whether release of tissue antigens from necrotic thyroid epithelial cells can trigger DC maturation and initiation of a primary anti-self response. DC were cocultured with either viable (VT/DC) or necrotic (NT/DC) thyrocytes, and their phenotypic and functional maturation as well as immunopathogenic potential were assessed. Significant up-regulation of surface MHC class II and costimulatory molecule expression was observed in NT/DC but not in VT/DC. This was correlated with a functional maturation of NT/DC, determined by IL-12 secretion. Challenge of CBA/J mice with NT/DC, but not with VT/DC, elicited thyroglobulin (Tg)-specific IgG as well as Tg-specific CD4(+) T-cell responses and led to development of experimental autoimmune thyroiditis. These results support the view that thyroid epithelial cell necrosis may cause autoimmune thyroiditis via maturation of intrathyroidal DC.

Induction of hyperthyroidism in mice by intradermal immunization with DNA encoding the thyrotropin receptor.

Intramuscular injection with plasmid DNA encoding the human thyrotropin receptor (TSHR) has been known to elicit symptoms of Graves' disease (GD) in outbred but not inbred mice. In this study, we have examined, firstly, whether intradermal (i.d.) injection of TSHR DNA can induce hyperthyroidism in BALB/c mice and, secondly, whether coinjection of TSHR- and cytokine-producing plasmids can influence the outcome of disease. Animals were i.d. challenged at 0, 3 and 6 weeks with TSHR DNA and the immune response was assessed at the end of the 8th or 10th week. In two experiments, a total of 10 (67%) of 15 mice developed TSHR-specific antibodies as assessed by flow cytometry. Of these, 4 (27%) mice had elevated thyroxine (TT4) levels and goitrous thyroids with activated follicular epithelial cells but no evidence of lymphocytic infiltration. At 10 weeks, thyroid-stimulating antibodies (TSAb) were detected in two out of the four hyperthyroid animals. Interestingly, in mice that received a coinjection of TSHR- and IL-2- or IL-4-producing plasmids, there was no production of TSAbs and no evidence of hyperthyroidism. On the other hand, coinjection of DNA plasmids encoding TSHR and IL-12 did not significantly enhance GD development since two out of seven animals became thyrotoxic, but had no goitre. These results demonstrate that i.d. delivery of human TSHR DNA can break tolerance and elicit GD in inbred mice. The data do not support the notion that TSAb production is Th2-dependent in murine GD but they also suggest that codelivery of TSHR and Th1-promoting IL-12 genes may not be sufficient to enhance disease incidence and/or severity in this model.

Assessment of the frequency of mutant (hprt-) T lymphocytes from peripheral blood of patients with Hashimoto's thyroiditis.

A salient feature of Hashimoto's thyroiditis (HT) is the T-cell-mediated destruction of the thyroid gland leading to hypothyroidism. In HT, as in other autoimmune diseases, a central premise has been that autoreactive T cells must be dividing in response to autoantigens, accumulating random spontaneous mutations during the activation process. Here, we have examined this hypothesis by using as monitor of somatic cell mutation the hprt gene, encoding the salvage pathway enzyme hypoxanthine-guanine phosphoribosyl transferase. Eleven newly diagnosed patients with HT and 10 patients with chronic disease were selected for the study, whereas 10 healthy individuals were used as controls. Peripheral T cells were cultured under limiting dilution conditions in the presence of 6-thioguanine and the frequency (MF) of surviving mutant hprt(-) T cells was calculated by Poisson statistics. It was observed that the mean MF value of either patient group (6.6 +/- 5.8 per 10(6) cells for the newly diagnosed, and 8.8 +/- 4.0 per 10(6) cells for the patients with chronic disease) was not significantly different (p > 0.05) from that of the control group (6.8 +/- 6.4 per 10(6) cells). These data do not support the concept that patients with HT have an increased number of actively dividing T cells in the circulation compared to healthy controls. Autoreactive T cells may be activated mainly in situ or home readily to the thyroid in the early stages of the disease and reach a nonexpansion stage as the chronic disease is stabilized.

Pathogenic thyroglobulin peptides as model antigens: insights on the induction and maintenance of autoimmune thyroiditis.

In recent years, the discovery of pathogenic thyroglobulin (Tg) peptides has given a new impetus to study, at the basic level, mechanisms of induction and immunoregulation of autoimmune thyroiditis. The genetic control of the immune response against defined Tg epitopes and the diversity of the T-cell receptor repertoire recruited for their recognition were among the first issues examined. Some of these epitopes contained hormonogenic sites, i.e. thyroxine residues, and thus offered an excellent opportunity to study how post-translational modifications such as iodination, can influence induction of thyroiditogenic cells. The delineation of pathogenic Tg determinants also enabled the search for "molecular mimics" i.e. peptides of microbial origin that may be involved in the pathogenesis of the disease. In addition, factors promoting the generation of pathogenic epitopes during Tg processing in antigen presenting cells could now be systematically investigated. This review summarizes recent findings in these areas.

Enhancing or suppressive effects of antibodies on processing of a pathogenic T cell epitope in thyroglobulin.

Thyroglobulin (Tg)-specific Abs occur commonly in thyroid disease, but it is not clear to what extent they affect Tg processing and presentation to T cells. Here we show that generation of the nondominant pathogenic Tg epitope (2549-2560), containing thyroxine (T4) at position 2553 (T4(2553)), is augmented by Tg-specific IgG mAbs that facilitate FcR-mediated internalization of Tg. However, other mAbs of the same (IgG1) subclass enhanced Tg uptake by APC but had no effect on the generation of this peptide. Treatment of APC with chloroquine or glutaraldehyde abrogated enhanced generation of T4(2553). The boosting effect was selective, since the enhancing mAbs did not facilitate generation of the neighboring cryptic (2495-2511) peptide, which is also pathogenic in mice. When Tg was simultaneously complexed to a mAb reactive with T4(2553) and to a mixture of boosting mAbs, the presentation of this epitope was totally suppressed. These results suggest that Tg-specific Abs alter Tg processing and may boost or suppress the presentation of nondominant pathogenic determinants during the course of disease.

Involvement of epitope mimicry in potentiation but not initiation of autoimmune disease.

We have examined whether the peptide (368-381) from the murine adenovirus type 1 E1B sequence, exhibiting a high degree of homology with the known pathogenic thyroglobulin (Tg) T cell epitope (2695-2706), can induce experimental autoimmune thyroiditis (EAT) in SJL/J mice. The viral peptide was a poor immunogen at the T or B cell level and did not elicit EAT either directly or by adoptive transfer assays. Surprisingly, however, the viral peptide was highly antigenic in vitro, activating a Tg2695-2706-specific T cell clone and reacting with serum IgG from mice primed with the Tg homologue. The viral peptide also induced strong recall responses in Tg2695-2706-primed lymph node cells, and subsequent adoptive transfer of these cells into naive mice led to development of highly significant EAT. These data demonstrate that nonimmunogenic viral peptides can act as agonists for preactivated autoreactive T cells and suggest that epitope mimicry may at times play a potentiating rather than a precipitating role in the pathogenesis of autoimmune disease.

Induction of thyroiditis in mice with thyrotropin receptor lacking serologically dominant regions.

Grave's disease (GD) is characterized by pathogenic autoantibodies to the human thyrotropin receptor (hTSH-R), and is frequently associated with a lymphocytic infiltrate of the thyroid gland. In attempts to establish a murine model of GD, we and others have previously shown that immunization of mice with recombinant preparations of the hTSH-R ectodomain induces high titres of specific antibodies, which, however, are not pathogenic, nor is the response accompanied by the development of thyroiditis. Since earlier reports identified the serological immunodominant determinants within the N- and C-terminal regions of hTSH-R ectodomain, we reasoned that immunization of mice with truncated fragments of ectodomain lacking these dominant regions might result in skewing of the response to other determinants of the molecule, with consequent induction of immunopathological features present in GD. We show here that multiple challenge of BALB/c mice with an amino acid fragment of residues 43-282 generates antibodies directed at hTSH-R peptides 37-56, 157-176, 217-236 and 232-251. This reactivity pattern is distinct from that induced previously with the whole ectodomain of hTSH-R in BALB/c animals. Thyroid function remained unaffected in these mice, suggesting that pathogenic antibodies were not being induced. Interestingly, some animals developed lymphocytic infiltration of the thyroid gland, clearly indicating the presence of pathogenic T cell determinants within the 43-282 fragment. Challenge with the related fragment 43-316 produced the same pattern of serological response to the synthetic peptides as fragment 43 282, but was not accompanied by thyroiditis. The results demonstrate: (i) the presence of thyroiditogenic determinants within hTSH-R, and (ii) that these pathogenic determinants are likely to be cryptic, as their effect is exhibited only when the hierarchy of immunodominance within hTSH-R is drastically altered.

Recruitment of multiple V beta genes in the TCR repertoire against a single pathogenic thyroglobulin epitope.

In autoimmune thyroid disease, the question whether thyroid-infiltrating, autoreactive T cells are derived from a polyclonal or oligoclonal subset has been the subject of considerable debate. In this report, we have examined the T-cell receptor (TCR) V beta profile of mouse clonal T cells responding to a single thyroiditogenic epitope, the As-restricted, 9mer mouse thyroglobulin (MTg) peptide (2496-04). In vitro recall assays based on lymph node cell (LNC) proliferation and cytokine release demonstrated that this peptide is a minimal T-cell epitope inducing a T-helper 1 (Th1) type of response in SJL hosts. A panel of cloned, interleukin-2 (IL-2)-secreting hybridomas was generated from this Th1 subset and their TCR-V beta gene utilization was assessed by reverse transcription-polymerase chain reaction (RT-PCR). Ten clones derived from two independent fusions were found to utilize three V beta gene families (V beta 2, 4, and 17). To the extent that Tg or other thyroid autoantigens encompass multiple pathogenic epitopes it appears unlikely from these data that a restricted TCR-V beta chain usage will be a general characteristic of thyroiditogenic T cells.

Suppression of murine thyroiditis via blockade of the CD40-CD40L interaction.

The CD40 ligand (gp39) is transiently expressed on activated CD4+ T cells and mediates cognate helper function by interacting with CD40 on B cells. Increasing evidence suggests, however, critical involvement of gp39 not only in antibody-mediated responses but also in the development of effector T cells. Here, we have investigated the effect of in vivo gp39 blockade on the induction of murine experimental autoimmune thyroiditis (EAT), a T-cell-mediated disease. Over a 5-week period, EAT was induced in SJL mice with thyroglobulin (Tg) and adjuvant. Concomitantly, mice received intraperitoneal (i.p.) injections of MR1, a gp39-specific hamster monoclonal antibody (mAb), at 4-day intervals. Control mice were challenged with Tg but received equivalent doses of hamster immunoglobulin (HIg). It was observed that the control mice developed severe thyroiditis whereas the MR1-treated mice exhibited very low levels of infiltration that were mostly focal in nature. Blockade of gp39 was effective since the Tg-specific IgG titres were low or undetectable in all MR1-treated animals compared with the controls. In addition, upon restimulation with Tg in vitro, lymph node cells (LNC) from Tg-primed, MR1-treated mice proliferated less strongly and secreted significantly lower amounts of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) than LNC from untreated or HIg-treated controls. These results strongly suggest that in vivo blockade of gp39 suppresses EAT by inhibiting the priming of inflammatory Tg-specific T-helper type 1 cells.

Searching for pathogenic epitopes in thyroglobulin: parameters and caveats.

Last year marked the 40th anniversary of the discovery that thyroglobulin (Tg) is a major autoantigen in autoimmune thyroiditis. The Tg molecule presents unique challenges for epitope mapping owing to its large size and extensive iodination. Consequently, pathogenic determinants have only recently been identified. Here, George Carayanniotis and Varada Rao summarize the approaches used to determine pathogenic Tg T-cell epitopes and discuss caveats in this unusual quest.

Contrasting immunopathogenic properties of highly homologous peptides from rat and human thyroglobulin.

The current lack of amino acid sequence data for mouse thyroglobulin (Tg) necessitates mapping of pathogenic T-cell epitopes on heterologous Tg in mouse experimental autoimmune thyroiditis (EAT). A prevailing assumption has been that epitopes sharing a high degree of amino acid homology among heterologous Tg are likely to exhibit the same immunopathogenic properties in the same host. In this report, we have examined this concept while working with the 18-mer rat(r)Tg(2695-13) peptide that was previously shown to elicit 'A'-restricted T cells and EAT in SJL mice. A major immunopathogenic T-cell epitope was localized within the 12-mer rTg(2695-06). It was found that the human 12-mer homologue that carries two Ser substitutions at Glu2703 and Thr2704 exhibited contrasting properties: it failed to activate Th1 cells in lymphokine and proliferation assays; it did not cross-react with rTg(2695-06) at the T-cell level; and it induced only focal thyroiditis following adoptive transfer of specific lymph node cells. These data highlight the caveat involved in extrapolating results of pathogenic T-cell epitope mapping across heterologous Tgs, even when such epitopes share a high degree of amino acid homology.

Unaltered thyroid function in mice responding to a highly immunogenic thyrotropin receptor: implications for the establishment of a mouse model for Graves' disease.

Grave's disease (GD) is a common disorder characterized by the presence of autoantibodies to the thyrotropin receptor. In the past, the exceedingly low expression of the thyrotropin receptor on thyrocytes has not allowed its purification in quantities sufficient to investigate the establishment of an animal model for this disease. In this study, we have purified the 398-amino acid, extracellular region of the human thyrotropin receptor (TSH-R.E) from insect cells using recombinant baculovirus, and explored its immunopathogenic properties in H-2b,d,q,k,s strains of mice. The receptor preparation was highly immunogenic since it elicited strong specific proliferative T cell responses as well as IgG responses in all strains tested. In addition, hyperimmunization with TSH-R.E induced (i) serum antibodies that blocked the binding of 125I-TSH to its receptor, a common feature of GD autoantibodies; and (ii) IgG that reacted with a synthetic peptide (residues 32-54) from the N-terminus of the receptor, a region implicated in the binding of thyroid stimulating antibodies. In SJL animals only, a weak antibody response to two other thyroid antigens, thyroglobulin and thyroid peroxidase, was also observed. The presence of these antibodies, however, was not accompanied by a detectable alteration in thyroid function as assessed by the measurement of serum TSH, T4 and iodine levels. Also mononuclear infiltration of the thyroid gland or morphological changes compatible with an activation state of thyrocytes were not apparent in TSH-R-challenged mice. In contrast, mice treated with the anti-oxidant aminotriazole showed a dramatic increase in serum TSH levels and an activated follicular epithelium. These data demonstrate that a highly immunogenic TSH-R.E in mice does not necessarily provide a proper stimulus for the induction of a hyper- or hypothyroid status as defined by hormonal or histological criteria. Main reasons for the inability to induce receptor-specific antibodies that affect thyroid function such as those generated in GD are likely to be the inappropriate folding of the recombinant extracellular domain of the receptor, or the xenogeneic nature of the autoantigen.

Autoreactive IgG elicited in mice by the non-dominant but pathogenic thyroglobulin peptide (2495-2511): implications for thyroid autoimmunity.

We have previously shown that mice challenged with the rat thyroglobulin (Tg) peptide TgP1 (corresponding to aa 2495-2511 of human Tg) develop experimental autoimmune thyroiditis (EAT) and produce IgG antibodies that cross-react with Tg from various species. It was not clear, however, whether such antibodies were TgP1-specific or were induced secondarily--i.e. by autologous Tg released from the destroyed gland--and therefore directed to determinants other than TgP1. In this study we describe that, 5 weeks after priming with TgP1, the binding of serum IgG on native Tg is completely inhibited by free peptide, suggesting lack of recognition of other determinants on mouse Tg (mTg). In addition, TgP1-induced but not mTg-induced IgG bound better to heat-denatured than intact mTg, a result compatible with the recognition of a linear epitope by the peptide-induced antibodies. Comparison of the IgG subclass distribution among mTg-induced versus TgP1-induced IgG did not reveal qualitative differences, since all subclasses were represented in the order IgG1 > IgG2b > IgG2a > IgG3. Finally, TgP1-specific IgG reacted strongly with the follicular colloid in sections of normal thyroids, indicating the potential to bind to native Tg in vivo. These data: (i) highlight TgP1 as the only, so far, Tg sequence known to generate both EAT and Tg-reactive IgG in mice; and (ii) do not provide evidence for an amplification of the Tg-specific IgG response through the involvement of endogenous autoantigen in EAT.

Mapping of thyroglobulin epitopes: presentation of a 9mer pathogenic peptide by different mouse MHC class II isotypes.

We have previously reported that the 17mer thyroglobulin (Tg) peptide TgP1 (a.a. 2495-2511) induces experimental autoimmune thyroiditis (EAT) in H-2k mice, a process requiring expression of Ek genes, and in H-2s mice that lack functional E molecules. To test whether this apparent discrepancy was due to recognition of distinct TgP1 determinants in each strain, we mapped in this study minimal T-cell epitopes within TgP1 and examined their pathogenicity in C3H (H-2k) or SJL (H-2s) mice. Truncation analysis using TgP1-specific, CD4+ hybridomas from C3H mice identified two overlapping determinants, (2496-2504) and (2499-2507), that were restricted by the Ek and Ak molecules, respectively. Subsequent challenge of C3H and SJL mice with these 9mer peptides revealed that the Ek-restricted (2496-2504) determinant elicited EAT and specific proliferative LNC responses in both strains, suggesting recognition in the context of As, since this is the only class II molecule expressed in SJL mice. This was further confirmed by blocking of the proliferative LNC response by an As-specific monoclonal antibody. In contrast, the Ak-restricted (2499-2507) determinant induced weak EAT and no proliferative LNC responses in either strain. These data 1) delineate the 9mer (2496-2504) peptide as a minimal Tg T-cell epitope with direct pathogenic potential in mice and 2) highlight the use of nonisotypic MHC class II molecules for the presentation of this peptide in mice of different H-2 haplotypes. T-cell level. Despite its nondominant nature, TgP1 induces experimental autoimmune thyroiditis (EAT) with a genetic pattern similar to that obtained with intact Tg, and elicits strong specific T-cell responses as well as IgG responses that cross-react with Tgs from different species (Chronopoulou et al. 1992). For these reasons, TgP1-mediated EAT provides an excellent model for studying the immunoregulatory mechanisms leading to mononuclear infiltration of the thyroid and hypothyroidism--the main symptoms of Hashimoto's disease in humans (Weetman 1992). Earlier work (Chronopoulou et al. 1993) has demonstrated that Ek expression is a necessary but not sufficient requirement for EAT induction with TgP1 and suggested involvement of the Kk and/or Ak loci in the disease process. In apparent contrast to these findings, however, SJL mice that lack H-2E molecules were found to be EAT-susceptible after TgP1 challenge (Chronopoulou et al. 1992).(ABSTRACT TRUNCATED AT 400 WORDS)

Distinct genetic pattern of mouse susceptibility to thyroiditis induced by a novel thyroglobulin peptide.

Experimental autoimmune thyroiditis (EAT), induced by thyroglobulin (Tg) and adjuvant, is major histocompatibility complex-controlled and dependent on Tg-reactive T cells, but the immunopathogenic T-cell epitopes on Tg remain mostly undefined. We report here the thyroiditogenicity of a novel rat Tg peptide (TgP2; corresponding to human Tg amino acids 2695-2713), identified by algorithms as a site of putative T-cell epitope(s). TgP2 causes EAT in SJL (H-2s) but not in C3H or B10.BR (H-2k), BALB/c (H-2d), and B10 (H-2b) mice. This reveals a new genetic pattern of EAT susceptibility, since H-2k mice are known to be high responders (susceptible) after Tg challenge. Following in vivo priming with TgP2, T cells from only SJL mice proliferated significantly and consistently to TgP2 in vitro, whereas TgP2-specific IgG was observed in all strains tested. Adoptive transfer of TgP2-primed SJL lymph node cells to naive syngeneic recipients induced a pronounced mononuclear infiltration of the thyroid, which was more extensive than that observed after direct peptide challenge. TgP2 is non-immunodominant, since priming of SJL mice with rTg did not consistently elicit T-cell responses to TgP2 in vitro and a TgP2-specific T-cell hybridoma did not respond to antigen presenting cells pulsed with rTg. The data support the notion that Tg epitopes need not be either iodinated or immunodominant in order to cause severe thyroiditis and that the genetic pattern of the disease they induce can be distinct from that of Tg-mediated EAT.

Immunotargeting of thyroglobulin on antigen presenting cells abrogates natural tolerance in the absence of adjuvant.

Mice usually develop strong IgG responses to self-thyroglobulin (Tg) following immunization with mouse Tg (mTg) emulsified in complete Freund's adjuvant (CFA). Here we report that adjuvant-free challenge of mice with small doses of mTg conjugated onto a monoclonal antibody (MAb) specific for class II MHC determinants (anti-I-Ak) induces an mTg-specific IgG response in CBA (H-2k) but not in B6 (H-2b) mice. This is not a result of nonspecific uptake of immunoconjugate or chemical modification of mTg because mTg conjugated in a similar manner to a control MAb (specific for influenza nucleoprotein) of the same IgG subclass as the anti-I-Ak MAb did not elicit an autoimmune response. Despite the presence of mTg-specific IgG with titers equal to those observed after challenge with mTg in CFA, thyroid lesions were not detected in CBA mice that received mTg-(anti-I-Ak Mab) conjugate indicating a clear divergence in the requirements for autoantibody production and disease. The data suggest that small amounts of soluble autoantigen, conjugated onto MAbs specific for determinants expressed on antigen-presenting cells (APC), can effectively abrogate natural tolerance perhaps via a targeting mechanism that focuses autoantigen on APC. This approach may help elucidate the role of various APC subsets in autoimmunity and allow the study of initial events that trigger autoreactivity outside a CFA-induced granuloma site.

Induction of experimental autoimmune thyroiditis in rats with the synthetic peptide (2495-2511) of thyroglobulin.

We have previously shown that the synthetic 17 mer peptide TgP1 (a.a. 2495-2511) from the rat thyroglobulin (Tg) sequence is thyroiditogenic in mice and consists of nonimmunodominant but highly immunogenic determinants at the T or B cell level. Although TgP1 appears to be phylogenetically conserved, it remains uncertain whether it is a self-peptide in mice since the mouse Tg amino acid sequence is unknown. In this report, we demonstrate that TgP1 also induces experimental autoimmune thyroiditis (EAT) in WKY, F344, and WF strains of rats that are known to be EAT-susceptible after Tg challenge. Priming of all strains with TgP1 led to strong proliferative lymph node cell responses to the peptide in vitro that were abrogated by MAbs against CD4, OX-6, and OX-17 determinants, suggesting TgP1 recognition by class II-restricted T cells. In vivo priming with autologous or heterologous Tg did not lead to TgP1-specific proliferation in vitro confirming the cryptic nature of this sequence in rats. In contrast to findings in the mouse, strong peptide-specific IgG responses were not observed in rats despite the presence of EAT. These data demonstrate that TgP1 is immunopathogenic in an autologous system and provide the first evidence of a defined autoantigenic Tg peptide in rats.

Identification of a thyroiditogenic sequence within the thyroglobulin molecule.

Thyroglobulin (Tg)-specific T cells are important in the induction of experimental autoimmune thyroiditis (EAT), but the nature and the number of the Tg T cell epitopes involved in the disease process are unknown. Through the use of computerized algorithms that search for putative T cell epitopes, a 17-mer peptide (TgP1) was identified within the known portion of the rat Tg sequence (corresponding to amino acids 2495 to 2511 of the human Tg sequence) that induced strong mononuclear cell infiltration of the thyroid in classic EAT-susceptible murine strains such as SJL, C3H, and B10.BR and low or undetectable infiltration in EAT-resistant strains such as BALB/c and B10. TgP1 appears to be phylogenetically conserved since it is completely homologous to its bovine counterpart and differs at a single amino acid position from its human analogue. After priming with TgP1 in vivo, significant proliferative T cell responses to TgP1 in vitro were observed only with lymphocytes from susceptible (high responder) strains, thus correlating proliferative capacity with EAT induction. TgP1-primed T cells did not respond to intact mouse Tg (MTg) or rat Tg in vitro and, conversely, T cells primed in vivo with MTg or rat Tg did not respond to TgP1 in culture, suggesting that TgP1 is comprised of non-immunodominant T cell determinants. TgP1 was defined as a serologically nonimmunodominant epitope as well, since in vivo priming of all strains with MTg led to strong MTg-specific IgG responses but no TgP1-specific responses in ELISA assays. This was not due to lack of immunogenic B cell determinants on TgP1, however, because peptide challenge of EAT-susceptible strains elicited TgP1-specific IgG that also cross-reacted with MTg and rat, human, bovine, and porcine Tg. The data demonstrate that TgP1 delineates nonimmunodominant but highly immunogenic determinants at both the T and B cell level, which may play an important role in the development of autoimmune thyroiditis.