Tg Peptides Research Articles

Effective Inhibition of Thyroid Antigen Presentation Using Retro-Inverso Peptides in Experimental Autoimmune Thyroiditis: A Pathway Toward Immune Therapies of Thyroid Autoimmunity.

Background: Autoimmune thyroid diseases (AITD) represent the most common autoimmune diseases. However, current therapies focus on relieving the symptoms instead of curing AITD, and new therapies to reverse the autoimmune attack on the thyroid are needed. HLA-DRβ1-Arg74 is the key HLA class II allele that triggers AITD by presenting pathogenic thyroglobulin (Tg) peptides that activate thyroid self-reactive T cells. We hypothesized that blocking the presentation of Tg peptides to T cells within the HLA-DRβ1-Arg74 peptide binding cleft could reverse the autoimmune response to the thyroid in AITD. Methods: The approach we used to block Tg peptide presentation within HLA-DRβ1-Arg74 is to design retro-inverso D-amino acid (RID) peptides that have high affinity to the HLA-DRβ1-Arg74 peptide binding pocket. Results: By using computational approaches and molecular dynamics simulations, we designed two RID peptides, RT-15 and VT-15, that blocked peptide binding to recombinant HLA-DRβ1-Arg74 molecule, as well as T cell activation in vitro. Furthermore, RT-15 and VT-15 blocked in vivo T cell activation by thyroglobulin in humanized NOD-DR3 mice induced with experimental autoimmune thyroiditis. Conclusions: In summary, we discovered two RID peptides that block thyroglobulin peptide binding to HLA-DRβ1-Arg74 and their presentation to T cells in AITD. These findings set the stage for a personalized medicine therapeutic approach for AITD patients who carry the DRβ1-Arg74 allele. This antigen-specific therapeutic strategy can potentially be extended to other autoimmune diseases.

A novel thrombin-based triagonist with diabetes-protective and weight-lowering potential

AimsTo investigate the diabetes-protective effect and weight-lowering potential of a novel long-acting triagonist at three metabolically related hormone receptors including glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), glucagon receptors. Main methodsTriagonist were designed in an iterative manner from native GLP-1, GIP and Glucogan. Main peptide chain (termed TG peptides) and subsequently modified LTG peptides were synthesized via solid phase synthesis. In vitro receptor activity assay was performed to screen the TG peptide with most balanced potency on all three receptors. The in vitro biological activities of modified TG peptides were further investigated by albumin-binding measurement and proteolytic cleavage test. Subsequently, oral glucose tolerance test (OGTT), pharmacokinetic test and chronic study were subjected to the acute and long-term efficacy evaluation of selected fusion peptide, LTG-6. Key findingsTG-8 exhibited equally aligned constituent efficacy and supraphysiological potency on corresponding receptor without cross-reactivity. Modified TG-8, termed LTG-6, exerted the great binding affinity for human serum albumin and the enhanced rational controlled-release of TG-8 in vitro. Further OGTT in different gene knockout mice and diabetic mice demonstrated the promising hypoglycemic and insulinotropic abilities of LTG-6. After long-term treatment for 8 weeks, LTG-6 was proved superior to co-agonists to decrease the body weight and %HbA1c, improve reverse dyslipidemia and glycemic control in the DIO models. SignificanceLTG-6, as a newly designed long-acting triagonist, holds potential to correct the obesity related metabolic disorders.

Identifying a Small Molecule Blocking Antigen Presentation in Autoimmune Thyroiditis

We previously showed that an HLA-DR variant containing arginine at position 74 of the DRβ1 chain (DRβ1-Arg74) is the specific HLA class II variant conferring risk for autoimmune thyroid diseases (AITD). We also identified 5 thyroglobulin (Tg) peptides that bound to DRβ1-Arg74. We hypothesized that blocking the binding of these peptides to DRβ1-Arg74 could block the continuous T-cell activation in thyroiditis needed to maintain the autoimmune response to the thyroid. The aim of the current study was to identify small molecules that can block T-cell activation by Tg peptides presented within DRβ1-Arg74 pockets. We screened a large and diverse library of compounds and identified one compound, cepharanthine that was able to block peptide binding to DRβ1-Arg74. We then showed that Tg.2098 is the dominant peptide when inducing experimental autoimmune thyroiditis (EAT) in NOD mice expressing human DRβ1-Arg74. Furthermore, cepharanthine blocked T-cell activation by thyroglobulin peptides, in particular Tg.2098 in mice that were induced with EAT. For the first time we identified a small molecule that can block Tg peptide binding and presentation to T-cells in autoimmune thyroiditis. If confirmed cepharanthine could potentially have a role in treating human AITD.

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.

A novel pathogenic peptide of thyroglobulin (2208-2227) induces autoreactive T-cell and B-cell responses in both high and low responder mouse strains.

Experimental autoimmune thyroiditis (EAT) is commonly induced by thyroglobulin (Tg) or Tg peptides in mice genetically susceptible to thyroiditis. In the present study, we investigated the immunogenic and pathogenic potential of a novel 20mer human Tg peptide, p2208 (amino acids 2208-2227), in mouse strains classified as low (LR) or high (HR) responders in EAT. The peptide was selected for its content in overlapping binding motifs for MHC class II products, associated with either resistance (A(b)), or susceptibility (A(s), E(k)) to EAT. We therefore immunized LR BALB/c (H-2(d)) and C57BL/6 (H-2(b)) strains, as well as HR CBA/J (H-2(k)) and SJL/J (H-2(s)) mice with 100 nmol of p2208 in adjuvant and collected their sera, lymph nodes and thyroid glands for further analysis. The p2208 peptide was found to contain B-cell and cryptic T-cell epitope(s) in two of the four strains examined, one LR and one HR. Specifically, it elicited direct EAT in C57BL/6 mice (two of seven mice, infiltration index 1-3), as well as in SJL/J mice (two of six mice, infiltration index 1-2). Such an EAT model could provide insights into the immunoregulatory cascades taking place in resistant hosts.

Fine epitope mapping within the pathogenic thyroglobulin peptide 2340–2359: minimal epitopes retaining antigenicity across various MHC haplotypes are not necessarily immunogenic

We have previously reported that the 20-mer peptide p2340 (amino acids 2340-2359), of human thyroglobulin (Tg) has the unique feature that it causes experimental autoimmune thyroiditis (EAT) in mouse strains bearing high-responder (HR) or low-responder (LR) MHC haplotypes in Tg-induced EAT. In this study, we have employed fine epitope mapping to examine whether this property of p2340 is the result of recognition of distinct or shared minimal T-cell epitopes in the context of HR or LR MHC class II molecules. Use of overlapping peptides showed that a core minimal 9-mer epitope (LTWVQTHIR, amino acids 2344-2352) was recognized by p2340-primed T cells from both HR (H2(k,s) ) and LR (H2(b,d) ) strains, whereas a second 9-mer epitope (HIRGFGGDP, amino acids 2350-2358) was antigenic only in H2(s) hosts. Truncation analysis of LTWVQTHIR and HIRGFGGDP peptides delineated them as the minimal epitopes recognized by p2340-primed T cells from the above strains. Subcutaneous challenge of all mouse strains with the 9-mer core peptide LTWVQTHIR in adjuvant elicited specific lymph node cell proliferative responses and mild EAT only in HR hosts, highlighting this sequence as a minimal pathogenic Tg peptide in EAT. The 9-mer peptide HIRGFGGDP was not found to be immunogenic in H2(s) hosts. These data demonstrate that minimal T-cell epitopes, defined as autoantigenic in hosts of various MHC haplotypes, are not intrinsically immunogenic. Activation of naive autoreactive T cells may require contributions from flanking residues within longer peptide sequences encompassing these epitopes.

Efficacy of HLA-DRB1∗03:01 and H2E transgenic mouse strains to correlate pathogenic thyroglobulin epitopes for autoimmune thyroiditis

Thyroglobulin (Tg), a homodimer of 660 kD comprising 2748 amino acids, is the largest autoantigen known. The prevalence of autoimmune thyroid disease, including Hashimoto’s thyroiditis and Graves’ disease, has provided the impetus for identifying pathogenic T cell epitopes from human Tg over two decades. With no known dominant epitopes, the search has long been a challenge for investigators. After identifying HLA-DRB1∗03:01 (HLA-DR3) and H2E b as susceptibility alleles for Tg-induced experimental autoimmune thyroiditis in transgenic mouse strains, we searched for naturally processed T cell epitopes with MHC class II-binding motif anchors and tested the selected peptides for pathogenicity in these mice. The thyroiditogenicity of one peptide, hTg2079, was confirmed in DR3 transgenic mice and corroborated in clinical studies. In H2E b-expressing transgenic mice, we identified three T cell epitopes from mouse Tg, mTg179, mTg409 and mTg2342, based on homology to epitopes hTg179, hTg410 and hTg2344, respectively, which we and others have found stimulatory or pathogenic in both DR3- and H2E-expressing mice. The high homology among these peptides with shared presentation by DR3, H2E b and H2E k molecules led us to examine the binding pocket residues of these class II molecules. Their similar binding characteristics help explain the pathogenic capacity of these T cell epitopes. Our approach of using appropriate human and murine MHC class II transgenic mice, combined with the synthesis and testing of potential pathogenic Tg peptides predicted from computational models of MHC-binding motifs, should continue to provide insights into human autoimmune thyroid disease.

Iodine content of thyroglobulin in NOD.H2h4 mice developing iodine-accelerated autoimmune thyroiditis

In NOD.H2h4 mice, high dietary iodine intake has been known to cause iodine-accelerated spontaneous autoimmune thyroiditis (ISAT) via an unknown mechanism. The aim of the study was to examine whether the NOD.H2h4 genetic background predisposes to enhanced iodine organification in thyroglobulin (Tg), a target autoantigen in ISAT. To avoid issues associated with an ongoing anti-Tg antibody response, we assessed Tg iodination levels in iodine-fed, B-cell deficient NOD.H2h4 mice. Additionally, we tested whether humoral or cellular immune responses of iodine-fed NOD.H2h4 mice are preferentially directed to Tg with increased iodine content (I-Tg) or known pathogenic Tg peptides that contained iodine. The iodine content of Tg was not significantly different between control (9.0 +/- 2.7 I atoms per monomer) and iodine-fed mice (10.9 +/- 0.3 I atoms per monomer). Furthermore, in iodine-fed NOD.H2h4 mice developing ISAT, strong but equivalent serum IgG responses were detected to both Tg or I-Tg, whereas their lymphoid cells were stimulated weakly but equally well by Tg or I-Tg in vitro and did not show reactivity against a panel of five pathogenic Tg peptides that contained iodine. The results suggest that development of ISAT in NOD.H2h4 mice is not associated with enhanced iodine organification or differential B- or T-cell responses to iodinated determinants in Tg.

Tg.2098 is a major human thyroglobulin T-cell epitope

An HLA-DR variant containing Arginine at position 74 of the DRβ1 chain confers a strong genetic susceptibility to autoimmune thyroid diseases (AITD), Graves' disease (GD) and Hashimoto's thyroiditis (HT), while Glutamine at position DRβ1–74 is protective. We hypothesized that the DRβ1-Arg74 variant is able to present pathogenic thyroglobulin (Tg) peptides to T-cells more efficiently, thereby triggering thyroid autoimmunity. Indeed, we have previously identified 4 human Tg (hTg) peptides that bind specifically to DRβ1-Arg74 with much weaker binding to the protective variant DRβ1-Gln74. The aim of our study was to examine in vivo whether an hTg peptide that binds strongly and specifically to DRβ1-Arg74 is capable of stimulating T-cells during the induction of thyroiditis in a “humanized” mouse expressing human DR3, and in patients positive for Tg antibodies. Sequencing of exon 2 of the DR transgene in the DR3 mice, null for endogenous MHC II molecules, confirmed that they expressed the disease-associated DRβ1-Arg74 variant, thus making them an ideal in vivo model to test the presentation of hTg peptides by DRβ1-Arg74 HLA-DR. Induction of EAT in the DR3 mice lead to T-cell stimulation and proliferation to Tg.2098, a strong and specific DRβ1-Arg74 binder, while a non-binding control peptide, Tg.2766 did not induce this response. Moreover, Tg.2098 stimulated T-cells from 4 individuals who were positive for thyroglobulin antibodies, demonstrating that Tg.2098 is an immunogenic peptide capable of being presented in vivo and activating T-cells in EAT and AITD. Energetic analysis of the complex formed by Tg.2098 and DRβ-Arg74 has shown that the origin of the affinity was determined by residues 1, 7 and 9 in the peptide, while the selectivity of the peptide for the MHC was determined by the Asp in position 4. The disease-protective substitution R74Q, leads to reduction in affinity due to changes in local interaction with D4 as well as non-local interaction with other residues. The electrostatic potential on the surface of the DRβ-Arg74–Tg.2098 complex has a unique signature which may be recognized by T-cell receptors leading to autoimmune thyroiditis. Taken together these findings suggest that Tg.2098, a strong and specific binder to the disease-associated HLA-DRβ-Arg74, is a major human T-cell epitope and participant in the pathoetiology of AITD.

Employing a Recombinant HLA-DR3 Expression System to Dissect Major Histocompatibility Complex II-Thyroglobulin Peptide Dynamism

Previously, we have shown that statistical synergism between amino acid variants in thyroglobulin (Tg) and specific HLA-DR3 pocket sequence signatures conferred a high risk for autoimmune thyroid disease (AITD). Therefore, we hypothesized that this statistical synergism mirrors a biochemical interaction between Tg peptides and HLA-DR3, which is key to the pathoetiology of AITD. To test this hypothesis, we designed a recombinant HLA-DR3 expression system that was used to express HLA-DR molecules harboring either AITD susceptibility or resistance DR pocket sequences. Next, we biochemically generated the potential Tg peptidic repertoire available to HLA-DR3 by separately treating 20 purified human thyroglobulin samples with cathepsins B, D, or L, lysosomal proteases that are involved in antigen processing and thyroid biology. Sequences of the cathepsin-generated peptides were then determined by matrix-assisted laser desorption ionization time-of-flight-mass spectroscopy, and algorithmic means were employed to identify putative AITD-susceptible HLA-DR3 binders. From four predicted peptides, we identified two novel peptides that bound strongly and specifically to both recombinant AITD-susceptible HLA-DR3 protein and HLA-DR3 molecules expressed on stably transfected cells. Intriguingly, the HLA-DR3-binding peptides we identified had a marked preference for the AITD-susceptibility DR signatures and not to those signatures that were AITD-protective. Structural analyses demonstrated the profound influence that the pocket signatures have on the interaction of HLA-DR molecules with Tg peptides. Our study suggests that interactions between Tg and discrete HLA-DR pocket signatures contribute to the initiation of AITD.

Application of HLA Class II Transgenic Mice to Study Autoimmune Regulation

In the past decade, we participated in the increased use of HLA class II transgenic mice to delineate genetic control in autoimmune diseases. Our studies began with individual class II transgenes to determine permissiveness for experimental autoimmune thyroiditis (EAT), first in resistant strains and then in the absence of endogenous H2 class II molecules. Polymorphism for HLA-DRB1 was observed, as DR3, but not DR2 or DR4, molecules serve as a determinant for EAT induction with either mouse thyroglobulin (mTg) or human thyroglobulin (hTg). This delineation enabled identification of pathogenic Tg peptides, based on DR3-binding motifs. HLA-DQ polymorphism was also detectable; hTg induced moderate EAT in DQ8(+), but not DQ6(+), mice. Coexpressing permissive and nonpermissive alleles, DR3(+) mice showed reduced EAT severity in the presence of DQ8, but not DQ6, DR2, or DR4. Determining the regulatory T cell (Treg) influences showed that Treg depletion increased thyroiditis incidence and severity without altering the major histocompatibility complex-based hierarchy in susceptibility. This increase after Treg depletion can also be observed in NaI-induced thyroiditis in DR3(+) mice, a means to study a major environmental factor in thyroid autoimmunity. DR3(+) mice were also immunized with human thyroid peroxidase cDNA, resulting in thyroiditis and an antibody (Ab) profile resembling patient antibodies (Abs). Similar immunization with human TSH receptor cDNA resulted in thyroid-stimulating Abs and elevated T(4) levels with moderate thyroiditis in some animals, suggesting a potential Graves' disease model that due to thyroid lesions is more complete than other models. Recently, Treg manipulation in cancer immunotherapy trials has triggered various autoimmune disorders. Thus, DR3(+) mice are being used to monitor a known risk factor for autoimmune thyroid disease in attempts to enhance tumor immunity.

Pathogenicity of a human thyroglobulin peptide (2340–2359) in mice with high or low genetic susceptibility to thyroiditis

We have previously identified a 20-mer peptide of human thyroglobulin (hTg), p2340 (aa2340-2359), which induced experimental autoimmune thyroiditis (EAT) in AKR/J (H-2(k)) and HLA-DR3 transgenic mice. In this study, we investigated the thyroiditogenic potential of p2340 in 'high responder' CBA/J (H-2(k)) and SJL/J (H-2(s)) or 'low responder' C57BL/6 (H-2(b)) and BALB/c (H-2(d)) mice. Mice were immunized subcutaneously with 100 nmol of p2340 in complete Freund's adjuvant (CFA) and both the proliferative capacity of their lymph node cells in the presence of p2340 or intact Tg and the production of peptide-specific antibodies were investigated. The p2340 peptide was found to contain B-cell and non-dominant T-cell epitope(s) in all strains tested. Moreover, it elicited EAT in CBA/J (2/6, infiltration index (I.I.) 1) and SJL/J (5/5, I.I. 1-3) mice after direct challenge and in BALB/c (4/7, I.I. 1) and C57BL/6 (1/5, I.I. 1) after adoptive transfer of p2340-primed lymph node cells. P2340 is the first Tg peptide found to be pathogenic in low as well as high responder mouse strains and thus will allow us to investigate mechanisms of EAT induction in a genetically resistant host.

Variable influences of iodine on the T‐cell recognition of a single thyroglobulin epitope

We have previously shown that iodotyrosyl formation within certain innocuous thyroglobulin (Tg) peptides confers on them immunopathogenic properties. In this report, we generated a panel of T-cell hybridoma clones specific for the immunogenic 16 mer Tg peptide p179 (amino acids 179-94) or its iodinated analogue (I-p179), with a view to examining the effects of a single iodine atom at the Y192 amino acid residue on T-cell recognition. We found that the peptide p179 was subdominant, and its binding to both A(k) and E(k) molecules was not significantly influenced by iodine. T-cell receptor (TCR) engagement was unaffected by the bulky iodine atom in two clones that responded to both analogues but it was sterically hindered in two other clones that recognized only p179. One clone was reactive only to I-p179, suggesting that the iodine atom is an integral part of its TCR ligand. Truncation analysis localized the determinant seen by all clones within the 11 mer peptide p184 (amino acids 184-194), suggesting that the cross-reactive clones were not activated by a minimal epitope lacking Y192 and that the negative influence of iodine was not the result of a flanking residue effect. These results demonstrate, at the clonal level, variable influences of a single iodine atom on the recognition of a single Tg peptide. Iodination of tyrosyl-containing, immunopathogenic Tg peptides may have unpredictable effects at the polyclonal level, depending on the extent of iodination at the particular site, and the relative number or effector function of autoreactive T-cell clones that are switched on or off by the neoantigenic determinant.

Modifying effects of iodine on the immunogenicity of thyroglobulin peptides

We have previously shown that iodotyrosyl formation within thyroglobulin (Tg) generates neoantigenic determinants that are immunopathogenic. In the current study, we have examined iodination effects on three tyrosyl-containing Tg peptides that are immunogenic in their non-iodinated form. We found that iodotyrosyl formation can enhance (p179, a.a. 179–194), suppress (p2540, a.a. 2540–2554), or not alter (p2529, a.a. 2529–2545) the immunogenic profiles of these peptides at the T-cell level. On the other hand, iodination did not alter the MHC-restriction profile of p2529 and p2540 (A k-binders) or p179 (A k- and E k-binder) and did not significantly influence the pathogenicity of these determinants. At the B-cell level, addition of an iodine atom on Y192 in p179 generated a neoantigenic determinant, but analogous effects were not discernible in p2529 or p2540. Our results demonstrate that iodotyrosyl formation can exert variable effects on the immunogenic behavior of Tg epitopes which may not always result in enhanced pathology. These findings also suggest that variations in the iodine content of Tg may significantly alter the hierarchy of antigenic determinants, to which the immune system may or may not be tolerant.

Iodination of Tyrosyls in Thyroglobulin Generates Neoantigenic Determinants That Cause Thyroiditis

Thyroglobulin (Tg) is unique in its ability to incorporate and store available iodine in the form of iodotyrosyl residues. Iodination of Tg has been known to increase its immunopathogenicity in experimental animals, presumably through the formation of iodine-containing neoantigenic determinants that can elicit an autoimmune response, but defined pathogenic Tg peptides carrying iodotyrosyls have not yet been identified. We report in this study that a systematic, algorithm-based search of mouse Tg has delineated three iodotyrosyl-containing peptides that activate autoreactive T cells and cause experimental autoimmune thyroiditis in normal CBA/J mice. These peptides (aa 117-132, 304-318, and 1931-1945) were not immunogenic in their native form, and iodination of tyrosyls facilitated either peptide binding to MHC or T cell recognition of the peptide. These results demonstrate that iodotyrosyl formation in normal Tg confers pathogenic potential to certain peptides that may otherwise remain innocuous and undetectable by conventional mapping methods.

Detection of thyroglobulin mRNA as truncated isoform(s) in mouse thymus

Recent studies employing reverse transription-polymerase chain reaction (RT-PCR) have demonstrated the intrathymic presence of mRNA for various autoantigens, including thyroglobulin (Tg). Deliberations on the mechanisms of central tolerance usually assume that this approach detects intact mRNA transcripts that can be translated to express the whole autoantigen in the thymus. In the present study, we tested this assumption using mRNA transcripts of mouse Tg which encode at least 13 pathogenic peptides, scattered over a large (8.5 kb) sequence. We found that mRNA encoding 11 out of these 13 Tg peptides was present in both the thyroid and the thymus of CBA/J mice, with no apparent temporal fluctuations in expression from birth to 12 weeks of age. Interestingly, detection of these sequences was also demonstrable in the liver and kidney, but not in muscle. However, mRNA encoding two pathogenic peptides (amino acids 1-12 and amino acids 1579-1591) was detected intrathyroidally but not in the other tissues. Further analysis by RT-PCR showed that Tg mRNA transcripts in the thymus, liver and kidney lack segments within the 1-915 bp and 961-5013 bp regions, spANNing exons 1-7 and 9-22, respectively. These data strongly suggest that certain known and perhaps other, as yet unmapped, pathogenic T-cell epitopes of Tg cANNot be encoded by the truncated isoform(s) of intrathymic Tg mRNA. These findings also imply that central tolerance to endogenous Tg produced by thymic epithelial cells may be incomplete.

Antibodies cross-reacting with thyroglobulin and thyroid peroxidase are induced by immunization of rabbits with an immunogenic thyroglobulin 20mer peptide

Thyroglobulin (Tg) and thyroid peroxidase (TPO) are two major autoantigens in autoimmune thyroid diseases (AITD). Cross-reactive anti-Tg/TPO antibodies have been identified in patients with AITD and in mice immunized with Tg or TPO. In the present study, we investigated the production of anti-Tg/TPO antibodies in rabbits immunized with human Tg and with a highly immunogenic Tg peptide (namely TgP41, sequence 2651-2670 of human Tg), by noncompetitive and competitive ELISA. TgP41 was found previously to induce intramolecular epitope spreading. We found that Tg-immunized rabbits developed a serological immune response to TPO due to cross-reactivity with Tg, since serum TPO reactivity was inhibited by soluble Tg and affinity-purified anti-Tg antibodies cross-reacted with TPO. Moreover, TgP41-immunized rabbits responded to Tg and TPO. This serological response was attributed to anti-Tg/TPO antibodies, based on the observation that serum TPO reactivity was again inhibited by soluble Tg, and affinity-purified anti-Tg antibodies, induced by TgP41-immunization, cross-reacted with TPO. Purified anti-TgP41 antibodies did not react with TPO, suggesting that a putative common antigenic determinant is not included in the peptide sequence. We propose that intermolecular spreading of reactivity to TPO observed after administration of the Tg-peptide is a result of intramolecular epitope spreading to determinant(s) responsible for Tg/TPO cross-reactivity.

Experimental Autoimmune Thyroiditis (EAT) Induced by the Thyroglobulin Peptide (2596–2608): Influence of H-2 and Non H-2 Genes

We have previously identified five thyroglobulin (Tg) peptides with Ak-binding motifs that induce experimental autoimmune thyroiditis (EAT) in CBA/J (H-2k) mice. In this study, we have examined whether H-2 or non H-2 genes can influence the immunopathogenicity of peptide p2596 (a.a. 2596–2608), which earlier elicited considerable pathology in CBA/J hosts. The p2596 peptide induced mild EAT—(infiltration index range=1–2)— in H-2-compatible AKR/J, B10.BR, and C3H/HeJ mice. Moreover, p2596-primed LNC from these mice exhibited peptide-specific proliferative responses and secreted significant amounts of IL-2 and IFN-γ in recall in vitro assays. Priming and boosting of these strains with p2596 resulted in the generation of specific IgG responses five weeks after the initial challenge. In contrast, s.c. challenge of H-2-incompatible strains such as DBA/1J (H-2q), SJL (H-2s), DBA/2J (H-2d) and C57BL/6 (H-2b) with the same peptide dose did not elicit EAT pathology and peptide-specific B- or T-cell responses. These data demonstrate the thyroiditogenic potential of p2596 in H-2k strains of diverse non-H-2 backgrounds but not in mice carrying H-2b, d, q or s haplotypes.

HLA and H2 Class II Transgenic Mouse Models to Study Susceptibility and Protection in Autoimmune Thyroid Disease

Using single H2 and HLA class II transgenic mice, in the absence of endogenous H2 class II molecules, we have studied the permissiveness of class II molecules for experimental autoimmune thyroiditis (EAT). Resistant strains expressing susceptible class II molecules, H2A k or HLA-DR3, developed EAT, clearly demonstrating the importance of class II gene inheritance. Polymorphism for HLA-DRB1 was observed, as DR3, but not DR2 or DR4, molecules were permissive for EAT induction with either mouse (m) or human (h) thyroglobulin (Tg). HLA-DQ polymorphism was also detectable, as hTg-induced EAT developed in DQ8+, but not DQ6+, mice. Class II gene interactions leading to reduced EAT severity were observed in H2 transgenic mice, when H2E transgene was expressed in H2A+ mice or H2A molecules were introduced into our novel H2A-E+ transgenic model. Similarly, in DR3+ mice, only the DQ8 transgene reduced EAT severity, depending on both background genes (C57BL/10 or NOD) and Tg species. Based on computer-predicted, class II-binding motifs, potential pathogenic Tg peptides, either unique to hTg (H2A-E+ model) or shared between mTg and hTg (HLA-DR3+ model), were identified. We have also developed a Graves' disease model by immunizing DR3+ mice with TSH receptor DNA. Thus, transgenic models are excellent tools to study human autoimmune thyroid diseases in the context of murine EAT.

Evidence for intramolecular B-cell epitope spreading during experimental immunization with an immunogenic thyroglobulin peptide.

Thyroglobulin (Tg) is a target autoantigen in autoimmune thyroid diseases, such as Graves' disease (GD) and Hashimoto's thyroiditis. In a previous study we identified three 20mer Tg peptides bearing epitopes of autoantibodies associated with GD (TgP15, TgP26 and TgP41: sequences 2339-2358, 2471-2490 and 2651-2670 of human Tg, respectively). In the present study, we investigated the antigenicity of the above peptides in experimental immunization with Tg, the immunogenicity of antigenic peptides and the possibility of intramolecular B-cell epitope spreading during peptide immunization. For this purpose, two rabbits were injected with human Tg in CFA six times, every three weeks. Two control animals were injected only with CFA. Testing of antisera and of affinity-purified antibodies, by ELISA against the three peptides, revealed reactivity only to TgP41. This synthetic peptide was subsequently administered to two rabbits, in its free form (100 micro g in CFA six times, every two weeks). A strong serological response was developed not only against TgP41, but also to intact human and rabbit Tg. Immunization with TgP41 induced intramolecular B-cell epitope spreading, i.e. production of antibodies to sites on Tg other than that corresponding to TgP41, as revealed by immunoadsorption and competitive ELISA. Histopathological studies did not reveal any infiltration in thyroid glands. We conclude that peptide TgP41 encompasses not only an epitope of disease-associated autoantibodies, but also a dominant immunogenic epitope of experimentally induced Tg-specific antibodies, able to drive B-cell epitope spreading.