Peptides of human thyroglobulin reactive with sera of patients with autoimmune thyroid disease

Autoantibodies to thyroglobulin (Tg) are a prominent feature of the two autoimmune thyroid diseases, chronic lymphocytic (Hashimoto's) thyroiditis and Graves' disease. Similar autoantibodies are found in the serum of many normal individuals without evidence of thyroid disease. Previous studies have indicated that patients with autoimmune thyroid disease recognize epitopes of Tg which are not usually recognized by normal individuals. The goal of this investigation was to identify peptide fragments of Tg bearing these disease-associated epitopes. For this purpose, we utilized a panel of mAbs that bind to different epitopes of the Tg molecule. One of these mAbs (137C1) reacted with an epitope that was also recognized by the sera of patients with autoimmune thyroiditis. In the present study, we show that two peptides (15 and 23 kDa) that reacted with mAb 137C1 are located in different parts of the Tg molecule. Each peptide inhibited the binding of mAb 137C1 to the other peptide and to the intact Tg, indicating that the same epitope was represented on the two peptides. Loops and helices of the secondary structure of the two peptides might be involved in the conformational epitope recognized by mAb 137C1. A striking finding of this study is that two apparently unrelated fragments of the Tg molecule bind to the same mAb. These findings may have important ramifications with regard to epitope spread and the progression of the autoimmune response to 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.     

Delineation of five thyroglobulin T cell epitopes with pathogenic potential in experimental autoimmune thyroiditis

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-2A(k) genes, we have used an algorithm-based approach to identify A(k)-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-2(k)) 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 A(k) binders because peptide-specific LNC proliferation was blocked by an A(k)-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 A(k)-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.     

Myelin-reactive type B T cells and T cells specific for low-affinity MHC-binding myelin peptides escape tolerance in HLA-DR transgenic mice

Genes of the MHC show the strongest genetic association with multiple sclerosis (MS), but the underlying mechanisms have remained unresolved. In this study, we asked whether the MS-associated MHC class II molecules, HLA-DRB1*1501, HLA-DRB5*0101, and HLA-DRB1*0401, contribute to autoimmune CNS demyelination by promoting pathogenic T cell responses to human myelin basic protein (hMBP), using three transgenic (Tg) mouse lines expressing these MHC molecules. Unexpectedly, profound T cell tolerance to the high-affinity MHC-binding hMBP82-100 epitope was observed in all Tg mouse lines. T cell tolerance to hMBP82-100 was abolished upon back-crossing the HLA-DR Tg mice to MBP-deficient mice. In contrast, T cell tolerance was incomplete for low-affinity MHC-binding hMBP epitopes. Furthermore, hMBP82-100-specific type B T cells escaped tolerance in HLA-DRB5*0101 Tg mice. Importantly, T cells specific for low-affinity MHC-binding hMBP epitopes and hMBP82-100-specific type B T cells were highly encephalitogenic. Collectively, the results show that MS-associated MHC class II molecules are highly efficient at inducing T cell tolerance to high-affinity MHC-binding epitope, whereas autoreactive T cells specific for the low-affinity MHC-binding epitopes and type B T cells can escape the induction of T cell tolerance and may promote MS.     

A self T cell epitope induces autoantibody response: mechanism for production of antibodies to diverse glomerular basement membrane antigens

The anti-glomerular basement membrane (GBM) Ab has been regarded as a prototypical example of pathogenic autoantibodies. However, the mechanism for elicitation of this Ab remains unknown. In the present paper, we report that the Ab to diverse GBM Ags was induced by a single nephritogenic T cell epitope in a rat model. The T cell epitope pCol(28-40) of noncollagen domain 1 of collagen type IV alpha3 chain not only uniformly induced severe glomerulonephritis but also elicited anti-GBM Ab in 76% of the immunized rats after prominent glomerular injury. Furthermore, we demonstrated that the anti-GBM Ab was not related to the peptidic B cell epitope nested in pCol(28-40); that is, 1) elimination of the B cell epitope, either by substitution of the critical residues of the B cell epitope or by truncation, failed to abrogate anti-GBM Ab production, and 2) the anti-GBM Ab, eluted from the diseased kidneys, reacted only with native GBM, but not with pCol(28-40). Confocal microscopy and immunoprecipitation further demonstrated that the eluted anti-GBM Ab recognized conformational B cell epitope(s) of multiple native GBM proteins. We conclude that autoantibody response to diverse native GBM Ags was induced by a single nephritogenic T cell epitope. Thus, anti-GBM Ab may actually be a consequence of T cell-mediated glomerulonephritis.     

Anti-idiotypic mimicry of a neutralizing epitope on the glycoprotein B complex of human cytomegalovirus.

Experiments were carried out to investigate the ability of rabbit anti-idiotype antibodies (Ab2), directed against an anti-human cytomegalovirus monoclonal antibody (Ab1), to induce neutralizing antibodies specific for the immunodominant glycoprotein B viral complex. Mice immunized with Ab2 produced anti-Ab2 (Ab3) that was both antigen and idiotype specific with regard to Ab1. We conclude that the Ab2 antibodies mimicked a neutralizing epitope and acted as a network antigen for inducing a specific anti-human cytomegalovirus antibody response in this experimental system.     

Demyelinating myelin oligodendrocyte glycoprotein-specific autoantibody response is focused on one dominant conformational epitope region in rodents

Conformational epitopes of myelin oligodendrocyte glycoprotein (MOG) provide a major target for demyelinating autoantibodies in experimental autoimmune encephalomyelitis and recent studies indicate that a similar situation may exist in multiple sclerosis. We recently solved the crystal structure of the extracellular domain of MOG (MOG(ex)) in complex with a Fab derived from the demyelinating mAb 8-18C5 and identified the conformational 8-18C5 epitope on MOG that is dominated by the surface exposed FG loop of MOG. To determine the importance of this epitope with regard to the polyclonal Ab response to MOG(ex) we investigated the effects of mutating His(103) and Ser(104), the two central amino acids of the FG loop, on Ab binding. Mutation of these two residues reduced binding of a panel of eight demyelinating conformation-dependent mAbs to <20% compared with binding to wild-type MOG(ex), whereas substitution of amino acids that do not contribute to the 8-18C5 epitope had only a minor effect on Ab binding. The same restriction was observed for the polyclonal MOG-specific Ab response of MOG DNA-vaccinated BALB/c and SJL/J mice. Our data demonstrate that the pathogenic anti-MOG Ab response primarily targets one immunodominant region centered at the FG loop of MOG. Comparison of the structure of MOG(ex) with the structures of related IgV-like domains yields a possible explanation for the focused Ab response.     

Autoantibodies to thyroglobulin are encoded by diverse V-gene segments and recognize restricted epitopes.

Murine experimental autoimmune thyroiditis has been used as a model for human autoimmune thyroiditis. Experimental autoimmune thyroiditis is induced in mice by immunization with mouse thyroglobulin (Tg) in CFA. To characterize the antibodies to this autoantigen, we have studied the binding specificities and determined the nucleotide sequences of monoclonal anti-Tg antibodies. The specificities of the mAb for determinants on Tg varied extensively. Seven of 16 mAb showed reactivity to only mTg, 4 reacted to Tg from more than one species and four reacted to a variety of Ag. Many of the mAb were competitively inhibited by thyroid hormones, suggesting that they recognize the hormonogenic sites on the Tg molecule. The mAb could be divided into at least seven reactivity patterns based on reciprocal competitive inhibition studies, indicating that mTg contains at least seven antigenic regions. DNA sequence analysis of the mAb showed that a large number of V region gene segments encoded the H and L chains. No evidence for preferential use of any V region family or gene segment was found. Gene segments from the VH 7183, Q52, J558, and VH10 families were used by heavy chains, and the V kappa 1, 4, 8, 9, 19, and 21 families were used by kappa-chains. The results indicate that the antigenic epitopes on mTg elicit a very diverse autoantibody response that is derived from a large number of V region gene segments. Many of these autoantibodies show specific reactivity with mTg indicating they recognize species specific epitopes. The results suggest that clonal deletion of autoreactive Ab to certain self-epitopes may not occur.     

A defect in deletion of nucleosome-specific autoimmune T cells in lupus-prone thymus: role of thymic dendritic cells

To study central tolerance to the major product of ongoing apoptosis in the thymus, we made new lines of transgenic (Tg) mice expressing TCR of a pathogenic autoantibody-inducing Th cell that was specific for nucleosomes and its histone peptide H4(71-94). In the lupus-prone (SWR x NZB)F1 (SNF1) thymus, introduction of the lupus TCR transgene caused no deletion, but marked down-regulation of the Tg TCR and up-regulation of endogenous TCRs. Paradoxically, autoimmune disease was suppressed in the alphabetaTCR Tg SNF1 mice with induction of highly potent regulatory T cells in the periphery. By contrast, in the MHC-matched, normal (SWR x B10. D2)F1 (SBF1), or in the normal SWR backgrounds, marked deletion of transgenic thymocytes occurred. Thymic lymphoid cells of the normal or lupus-prone mice were equally susceptible to deletion by anti-CD3 Ab or irradiation. However, in the steady state, spontaneous presentation of naturally processed peptides related to the nucleosomal autoepitope was markedly greater by thymic dendritic cells (DC) from normal mice than that from lupus mice. Unmanipulated thymic DC of SNF1 mice expressed lesser amounts of MHC class II and costimulatory molecules than their normal counterparts. These results indicate that apoptotic nucleosomal autoepitopes are naturally processed and presented to developing thymocytes, and a relative deficiency in the natural display of nucleosomal autoepitopes by thymic DC occurs in lupus-prone SNF1 mice.     

Thyroxine-binding antibodies inhibit T cell recognition of a pathogenic thyroglobulin epitope

Thyroid hormone-binding (THB) Abs are frequently detected in autoimmune thyroid disorders but it is unknown whether they can exert immunoregulatory effects. We report that a THB mAb recognizing the 5' iodine atom of the outer phenolic ring of thyroxine (T4) can block T cell recognition of the pathogenic thyroglobulin (Tg) peptide (2549-2560) that contains T4 at aa position 2553 (T4(2553)). Following peptide binding to the MHC groove, the THB mAb inhibited activation of the A(k)-restricted, T4(2553)-specific, mouse T cell hybridoma clone 3.47, which does not recognize other T4-containing epitopes or noniodinated peptide analogues. Addition of the same THB mAb to T4(2553)-pulsed splenocytes largely inhibited specific activation of T4(2553)-primed lymph node cells and significantly reduced their capacity to adoptively transfer thyroiditis to naive CBA/J mice. These data demonstrate that some THB Abs can block recognition of iodine-containing Tg epitopes by autoaggressive T cells and support the view that such Abs may influence the development or maintenance of thyroid disease.     

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.     

The PGK epitope of human thyroglobulin: A molecular marker of alternatively spliced thyroglobulin molecules?

Summary Human thyroglobulin (hTg) is the 2748 aa precursor of the thyroid hormones T3 and T4. In autoimmune thyroid diseases, autoantibodies to hTg appeared which showed a restricted epitope specificity for the central region of the molecule (residues 1149–1251). Our hypothesis to explain why this particular region becomes autoantigenic is presented, which involves the existence of truncated, alternatively spliced forms of hTg in the bloodstream. To try to prove this hypothesis, we have undertaken the identification of the peptide epitopes recognized by monoclonal antibodies on the thyroglobulin molecule by multiple peptide synthesis methods; we report here on the identification of the three-residue epitope, Pro-Gly-Lys in position 1282–1284 of the hTg sequence which is recognized by monoclonal antibodies Tg2 and Tg8. Due to their sequence specificity, these antibodies could provide a means to tag the region of the hTg sequence which is suggested to be the site of an alternative processing phenomenon. Our results are discussed in terms of both the specificity of anti-hTg monoclonal antibodies and of the mechanism of appearance of autoantibodies recognizing the central region of hTg.     

Angiotensin II (AII)-related idiotypic network. I. Common occurrence of AII internal image on rabbit anti-idiotypic antibodies

Internal images of foreign Ag have been demonstrated in a variety of systems as anticipated by the idiotypic network theory formulated by Jerne. However, they seem to be of rare occurrence. In order to estimate the actual frequency of antibodies bearing internal images (Ab2-beta) of angiotensin II (AII), a phylogenetically conserved peptide made up of eight amino acids, nine rabbits were immunized with affinity or protein A purified anti-AII antibodies (Ab1) from allotype-matched rabbits. Four of nine antiidiotypic antibodies (Ab2) exhibited internal image-like reactivity. They recognized all the polyclonal Ab1 tested, whatever the species (rabbit, mouse, guinea pig). In addition, they were strongly reactive with three mAb specific for a carboxy terminus epitope on AII (mAb 110, 199, and 211) and with a fourth monoclonal Ab1 (133) identifying a more central epitope. Advantage was taken of this reactivity with mAb1 to purify Ab2-beta by affinity chromatography of Ab2 on Sepharose 4B covalently linked to the three monoclonal Ab1 specific for the carboxy terminus epitope. The eluate displayed typical internal image properties: 1) it reacted with all the polyclonal Ab1 tested, 2) this reaction was completely abolished by AII, and 3) rabbits and mice immunized with the eluate all produced Ab1. The AII related idiotypic network is thus characterized by high frequency and immunogenicity of AII internal images. In addition, reactivity of the latter with monoclonal Ab1 indicates variable expression on Ab2-beta of the epitopes defined by the mAb on the nominal Ag.     

Protection from experimental autoimmune thyroiditis conferred by a monoclonal antibody to T cell receptor from a cytotoxic hybridoma specific for thyroglobulin.

A clonotypic mAb, AG7, has been prepared from splenocytes of CBA/J mice immunized with a cytotoxic T cell hybridoma, HTC2, specific for a pathogenic epitope of the thyroglobulin molecule in association with class I MHC Ag. AG7 binds to HTC2 cells but not to the other T cell hybridomas tested. Moreover, when used in functional studies, AG7 blocks the HTC2 capacity of specific target lysis. It also reacts with a determinant that comodulates with the CD3 Ag present on the surface of HTC2 cells. Immunoprecipitation of 125I-labeled solubilized HTC2 membranes demonstrated two bands located at 90 and 72 kDa under nonreducing conditions, which became a 46-kDa band under reducing conditions. Finally, when AG7 is injected into CBA/J mice, on day -1 before immunization with the pathogenic tryptic fragments of the thyroglobulin molecule, experimental autoimmune thyroiditis is abrogated. Thus, one of the multiple potential mechanisms of the protective immunity against EAT induced by HTC2 cells that we previously proposed, i.e., the generation of anti-clonotypic antibodies to HTC2 TCR, seems apparent.     

Maternal autoantibody triggers de novo T cell-mediated neonatal autoimmune disease

Although human maternal autoantibodies may transfer transient manifestation of autoimmune disease to their progeny, some neonatal autoimmune diseases can progress, leading to the loss of tissue structure and function. In this study we document that murine maternal autoantibody transmitted to progeny can trigger de novo neonatal pathogenic autoreactive T cell response and T cell-mediated organ-specific autoimmune disease. Autoantibody to a zona pellucida 3 (ZP3) epitope was found to induce autoimmune ovarian disease (AOD) and premature ovarian failure in neonatal, but not adult, mice. Neonatal AOD did not occur in T cell-deficient pups, and the ovarian pathology was transferable by CD4(+) T cells from diseased donors. Interestingly, neonatal AOD occurred only in pups exposed to ZP3 autoantibody from neonatal days 1-5, but not from day 7 or day 9. The disease susceptibility neonatal time window was not related to a propensity of neonatal ovaries to autoimmune inflammation, and it was not affected by infusion of functional adult CD4(+)CD25(+) T cells. However, resistance to neonatal AOD in 9-day-old mice was abrogated by CD4(+)CD25(+) T cell depletion. Finally, neonatal AOD was blocked by Ab to IgG-FcR, and interestingly, the disease was not elicited by autoantibody to a second, independent native ZP3 B cell epitope. Therefore, a new mechanism of neonatal autoimmunity is presented in which epitope-specific autoantibody stimulates de novo autoimmune pathogenic CD4(+) T cell response.     

Cutting edge: the conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class II molecule

Rheumatoid arthritis (RA) is genetically associated with MHC class II molecules that contain the shared epitope. These MHC molecules may participate in disease pathogenesis by selectively binding arthritogenic peptides for presentation to autoreactive CD4(+) T cells. The nature of the arthritogenic Ag is not known, but recent work has identified posttranslationally modified proteins containing citrulline (deiminated arginine) as specific targets of the IgG Ab response in RA patients. To understand how citrulline might evoke an autoimmune reaction, we have studied T cell responses to citrulline-containing peptides in HLA-DRB1*0401 transgenic (DR4-IE tg) mice. In this study, we demonstrate that the conversion of arginine to citrulline at the peptide side-chain position interacting with the shared epitope significantly increases peptide-MHC affinity and leads to the activation CD4(+) T cells in DR4-IE tg mice. These results reveal how DRB1 alleles with the shared epitope could initiate an autoimmune response to citrullinated self-Ags in RA patients.     

Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoafffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freund's complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freund's adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector. Received: 27 December 1999 / Accepted: 27 January 2000     

Detection of a regulatory idiotype on a spontaneous neonatal self-reactive hybridoma antibody

To investigate the physiologic relevance of an idiotype-driven regulation of the immune system, we began a search for spontaneous self-reactive hybridomas in neonatal BALB/c mice. We sought hybridoma antibodies reactive with thyroglobulin (Tg) and expressing Id62, a recurrent idiotype with regulatory properties borne on induced adult autoantibodies to Tg. We describe herein such a neonatal Tg binding/Id62-positive monoclonal antibody (mAb) B10H2, and compare it with prototype mAb 62, an adult Tg-binding/Id62-positive mAb. Both mAb react with the same Tg epitope, as demonstrated by their abilities to totally inhibit the binding of the other to Tg. Moreover, as assessed by a double-reciprocal plot of their binding to Tg, their relative affinities for Tg are comparable. Likewise, mAb B10H2 appears idiotypically similar to mAb 62 because it totally inhibits the binding of mAb 62 to homologous anti-idiotype. Finally, as previously shown for mAb 62, mAb B10H2 expresses Id62 independently on both heavy (H) and light (L) chains, as evidenced by the immunoblot binding of a specific anti-Id62 probe to separated H and L chains. By molecular genetic analysis both antibodies appear to have made use of a member of the same VH 7183 gene family. Altogether, these findings suggest that neonatal mAb B10H2 and adult mAb 62 are very similar if not identical, and regulatory idiotype Id62 may be germline encoded. Furthermore this observation supports, in general, the concept of an idiotype-driven regulation for autoimmune responses.     

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) inH-2 ^k mice, a process requiring expression ofE ^k genes, and inH-2 ^S 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-2 ^k) or SJL (H-2 ^S) 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 E^k and A^k molecules, respectively. Subsequent challenge of C3H and SJL mice with these 9mer peptides revealed that the Ekrestricted (2496–2504) determinant elicited EAT and specific proliferative LNC responses in both strains, suggesting recognition in the context of A^s, 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 A^k-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 differentH-2 haplotypes.     

High Titers of Autoantibodies to Glutamate Decarboxylase in Type 1 Diabetes Patients: Epitope Analysis and Inhibition of Enzyme Activity

ObjectiveAutoantibodies to glutamate decarboxylase (GAD65Ab) are found in patients with autoimmune neurological disorders or type 1 diabetes. The correct diagnosis of GAD65Ab-associated neurological disorders is often delayed by the variability of symptoms and a lack of diagnostic markers. We hypothesized that the frequency of neurological disorders with high GAD65Ab titers is significantly higher than currently recognized.MethodsWe analyzed GAD65Ab titer, GAD65 enzyme activity inhibition, and GAD65Ab epitope pattern in a cohort of type 1 diabetes patients (n = 100) and correlated our findings with neurological symptoms and diseases.ResultsOverall, 43% (43/100) of patients had detectable GAD65Ab titers (median = 400 U/mL, range: 142250,000 U/mL). The GAD65Ab titers in 10 type 1 diabetes patients exceeded the 90^th percentile of the cohort (2,000250,000 U/mL). Sera of these 10 patients were analyzed for their GAD65Ab epitope specificity and their ability to inhibit GAD65 enzyme activity in vitro. GAD65Ab of 5 patients inhibited the enzyme activity significantly (by 34-55%). Three patients complained of muscle stiffness and pain, which was documented in 2 of these patients.ConclusionsBased on our findings, we suggest that neurological disorders with high GAD65Ab titers are more frequent in type 1 diabetes patients than currently recognized. (Endocr Pract. 2013;19:663-668)