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Basil Rapoport

Publications and source records attributed to Basil Rapoport.

At least 19 recordsLinked to original sources
PubMed2006

Probing the genetic basis for thyrotropin receptor antibodies and hyperthyroidism in immunized CXB recombinant inbred mice.

Immunization with adenovirus encoding the TSH receptor (TSHR) or its A-subunit induces Graves' hyperthyroidism in BALB/c and BALB/c x C57BL/6 offspring but not C57BL/6 mice. High-resolution genetic maps are available for 13 recombinant inbred CXB strains generated from BALB/c x C57BL/6 progeny by repeated brother x sister matings to establish fully inbred lines. CXB strains were studied before and after A-subunit adenovirus immunization for TSHR antibodies (TBI, inhibition of TSH binding), serum T4, and thyroid histology. All strains developed TBI activity (at variable levels), six strains became hyperthyroid, and one was overtly thyrotoxic. No low TBI responders became hyperthyroid, but high TBI did not predict hyperthyroidism. Preimmunization T4 levels varied in different CXB strains and was unrelated to subsequent T4 elevation. Linkage analysis indicated that different chromosomes were involved in generating TSHR antibodies and serum T4 before and after immunization. TBI activity was linked in part with major histocompatibility (MHC) genes on chromosome 17 (Chr 17) but induced Graves' disease involved non-MHC genes (Chr 19 and 10). The Chr 10 locus is close to the Trhde gene that encodes TSH-releasing hormone degrading enzyme. Expression of Trhde is controlled by thyroid hormones and linkage with a thyroid function-related gene is intriguing. Our data, the first genome scan in murine Graves' disease, provides insight into the role of MHC and non-MHC genes in human and murine Graves' disease. Finally, our study demonstrates the potential of recombinant inbred mice for discriminating between immune-response genes and thyroid function susceptibility genes in Graves' disease.

Adenoviridae↗
PubMed2006

Evidence that human thyroid cells express uncleaved, single-chain thyrotropin receptors on their surface.

The prevailing concept is that, in human thyroid tissue in vivo, all cell-surface TSH receptors (TSHR) cleave into disulfide linked A and B subunits. Because this viewpoint is based on studies using homogenized thyroid tissue and because of TSHR fragility, we studied TSHR subunit structure in intact thyroid cells, primary human thyrocyte cultures, FRTL-5 rat thyroid cells, and WRO (follicular) and NPA (papillary) thyroid cancer cell lines. To overcome the handicap of very low TSHR expression in thyroid cells, we generated a TSHR-expressing adenovirus (TSHR-Ad-RGD) with an integrin-binding RGD motif enabling efficient entry into cells lacking the coxsackie-adenovirus receptor. Two days after TSHR-Ad-RGD infection, [125I]TSH cross-linking to intact cells revealed uncleaved, single-chain TSHR as well as cleaved TSHR A subunits on the surface of all four thyroid cell types. The extent of TSHR cleavage, which is independent of the level of TSHR expression, was consistently lower in the human thyroid cancer cell lines than in the other cell lines. In flow cytometry studies after TSHR-Ad-RGD infection, strong signals were detected in all four thyroid cell types using a monoclonal antibody that primarily recognizes the uncleaved TSHR. Finally, using the same monoclonal antibody, confocal microscopy confirmed the presence of single-chain TSHR on TSHR-Ad-RGD-infected thyroid cells. In summary, TSH covalent cross-linking, flow cytometry, and confocal microscopy demonstrate the presence of uncleaved TSHR on the human thyrocyte surface. These data provide stronger evidence for this alternative than the contrary view based on the finding of only cleaved TSHR in homogenized thyroid cells.

Animals↗
PubMed2006

Targeted expression of the human thyrotropin receptor A-subunit to the mouse thyroid: insight into overcoming the lack of response to A-subunit adenovirus immunization.

The thyrotropin receptor (TSHR), the major autoantigen in Graves' disease, is posttranslationally modified by intramolecular cleavage to form disulfide-linked A- and B-subunits. Because Graves' hyperthyroidism is preferentially induced in BALB/c mice using adenovirus encoding the free A-subunit rather than full-length human TSHR, the shed A-subunit appears to drive the disease-associated autoimmune response. To further investigate this phenomenon, we generated transgenic mice with the human A-subunit targeted to the thyroid. Founder transgenic mice had normal thyroid function and were backcrossed to BALB/c. The A-subunit mRNA expression was confirmed in thyroid tissue. Unlike wild-type littermates, transgenic mice immunized with low-dose A-subunit adenovirus failed to develop TSHR Abs, hyperthyroidism, or splenocyte responses to TSHR Ag. Conventional immunization with A-subunit protein and adjuvants induced TSHR Abs lacking the characteristics of human autoantibodies. Unresponsiveness was partially overcome using high-dose, full-length human TSHR adenovirus. Although of low titer, these induced Abs recognized the N terminus of the A-subunit, and splenocytes responded to A-subunit peptides. Therefore, "non-self" regions in the B-subunit did not contribute to inducing responses. Indeed, transgenic mice immunized with high-dose A-subunit adenovirus developed TSHR Abs with thyrotropin-binding inhibitory activity, although at lower titers than wild-type littermates, suggesting down-regulation in the transgenic mice. In conclusion, in mice expressing a human A-subunit transgene in the thyroid, non-self human B-subunit epitopes are not necessary to induce responses to the A-subunit. Our findings raise the possibility that autoimmunity to the TSHR in humans may not involve epitopes on a cross-reacting protein, but rather, strong adjuvant signals provided in bystander immune responses.

Adenoviridae↗
PubMed2006

HLA-DR3 transgenic mice immunized with adenovirus encoding the thyrotropin receptor: T cell epitopes and functional analysis of the CD40 Graves' polymorphism.

The major histocompatibility (MHC) molecule HLA-DR3 is a susceptibility gene for Graves' disease (GD) in Caucasians. Mice lacking murine MHC and expressing human HLA-DR3 develop thyrotropin receptor (TSHR) antibodies and sometimes hyperthyroidism after vaccination with TSHR-DNA. MHC molecules present peptides processed from antigens to T cells. Therefore, we used DR3-transgenic mice to investigate recognition of TSHR ectodomain peptides. After immunization with TSHR A-subunit adenovirus (A-subunit-Ad) but not control-adenovirus (Control-Ad), splenocytes from DR3 mice responded to A-subunit protein in culture by producing interferon-gamma (IFN-gamma). When challenged with 29 overlapping TSHR peptides, splenocytes from A-subunit-Ad- or Control-Ad-immunized mice responded to several peptides. However, in splenocytes from A-subunit-Ad but not Control-Ad mice, a peptide containing TSHR residues 142-161 induced significantly more IFN-gamma than the same splenocytes in medium alone. Immunized DR3 mice also permitted testing the TSHR-specific function of the CD40 single nucleotide polymorphism (C vs. T) associated with GD. Of three human DR3 human Epstein-Barr virus lines (EBVL), two had C in both alleles (CC) and one was CT. However, these EBVL presented peptides poorly and there was no difference between CC vs. CT EBVL in peptide presentation to splenocytes from immunized mice. A peptide corresponding to residues 145-163 is one of seven suggested to be important in GD based on HLA-binding affinities, T-epitope algorithms, and human studies. Consequently, as in human GD, TSHR amino acids 142-161 appear to include a major T cell epitope in HLA-DR3 transgenic mice immunized with A-subunit-Ad.

Adenoviridae↗
PubMed2006

Antibodies focused on the human autoantibody immunodominant region are induced by B lymphocytes that constitutively express thyroid peroxidase diverted to the major histocompatibility complex II pathway.

We addressed the question of why naturally occurring, polyclonal thyroid peroxidase (TPO) autoantibodies have a restricted epitopic repertoire to an immunodominant region (IDR). We hypothesized that immunizing BALB/c mice with major histocompatibililty complex (MHC) class II compatible B lymphocytes (A20 cells) preferentially diverting TPO to the MHC class II pathway would produce TPO antibodies with an epitopic specificity similar to human autoantibodies, namely to the IDR. For this purpose we stably expressed in A20 cells a fusion protein of TPO sandwiched between the signal peptide and transmembrane/cytoplasmic tail of the lysosome- associated membrane protein (LAMP) 1. Expression of LAMP1-TPO in A20 B cells was confirmed by flow cytometry using a TPO monoclonal antibody. Mice injected intraperitoneally with LAMP1-TPO A20 B cells developed TPO antibodies detected by enzyme-linked immunosorbent assay (ELISA), flow cytometry and (125)I-TPO precipitation. However, TPO antibody levels were low. Most important, competition for TPO antibody binding by recombinant human TPO autoantibody Fab indicated that more than 80% of the polyclonal TPO antibodies in the immunized mice were to the human autoantibody IDR. In summary, injecting mice with B lymphocytes that constitutively express TPO diverted to the MHC class II pathway generates antibodies with epitopes similar to those of human TPO autoantibodies, namely to the autoantibody IDR. However, these antibodies are of low titer that is itself associated with this epitopic bias.

Animals↗
PubMed2006

Blockade of costimulation between T cells and antigen-presenting cells: an approach to suppress murine Graves' disease induced using thyrotropin receptor-expressing adenovirus.

OBJECTIVE: Immune responses require costimulatory interactions between molecules on antigen-presenting cells and T cells: CD40 binding to CD40 ligand and B7 binding to CD28. Graves' hyperthyroidism is induced in BALB/c mice by immunization with thyrotropin receptor (TSHR) A-subunit adenovirus (Ad-A-subunit). We attempted to modulate Ad-A-subunit-induced Graves' disease using adenoviruses expressing costimulation "decoys": CD40-IgG-Fc (CD40-Ig) to block CD40:CD40-ligand interactions and CTLA4-Fc (CTLA4-Ig) to prevent B7:CD28 binding. OUTCOME: Unexpectedly, coimmunizing mice with Ad-A-subunit and excess control adenovirus (1:10 Ad-A-subunit:Ad-control) reduced TSHR antibody levels (thyrotropin binding inhibition [TBI]). Furthermore, only 15% of mice developed hyperthyroidism versus 75% using the same Ad-A-subunit dose (10(8) particles) without Ad-control. This effect was related to the dose of control adenovirus but not to the adenovirus insert, the timing or immunization site. Increasing the Ad-subunit dose (10(9) particles) and decreasing the control adenovirus dose (10:1 Ad-A-subunit:Ad-control) induced high TBI levels and 80% of mice were hyperthyroid. Coimmunization with Ad-CD40-Ig (but not Ad-CTLA4-Ig) reduced the incidence of hyperthyroidism to 40%. CONCLUSIONS: Using appropriate controls and adenovirus ratios, our data suggest the importance of CD40:CD40-ligand interactions for inducing Graves' hyperthyroidism by Ad-A-subunit. Furthermore, our observations emphasize the potential pitfalls of non-specific inhibition by coimmunization with two adenovirus species.

Adenoviridae↗
PubMed2005

Relationship between thyroid peroxidase T cell epitope restriction and antibody recognition of the autoantibody immunodominant region in human leukocyte antigen DR3 transgenic mice.

We investigated the relationship between thyroid peroxidase (TPO) antibody and T lymphocyte epitopes in TPO-adenovirus (TPO-Ad) immunized BALB/c mice and mice transgenic for the human class II molecule DR3 associated with human thyroid autoimmunity. TPO autoantibodies are largely restricted to an immunodominant region (IDR). BALB/c mice immunized with fewer (10(7) vs. 10(9)) TPO-Ad particles developed TPO antibodies with lower titers that displayed greater restriction to the IDR. However, as with higher-dose TPO-Ad immunization, T cell epitopes (assessed by splenocyte interferon-gamma response to TPO in vitro) were highly diverse and variable in different animals. In contrast, DR3 mice immunized the higher TPO-Ad dose (10(9) particles) had high TPO antibody levels that showed relative focus on the IDR. Moreover, T cell epitopes recognized by splenocytes from DR3 mice showed greater restriction than BALB/c mice. Antibody affinities for TPO were higher in DR3 than in BALB/c mice. The present study indicates that weak TPO-Ad immunization of BALB/c mice (with consequent low TPO antibody titers) is required for enhanced IDR focus yet is not associated with T cell epitopic restriction. Humanized DR3 transgenic mice, despite stronger TPO-Ad immunization, develop higher titer TPO antibodies that do focus on the autoantibody IDR with T cells that recognize a more limited range of TPO peptides. These data suggest a relationship between major histocompatibility complex class II molecules and the development of antibodies to the IDR, a feature of human thyroid autoimmunity.

Adenoviridae↗
PubMed2005

Insight into Graves' hyperthyroidism from animal models.

Graves' hyperthyroidism can be induced in mice or hamsters by novel approaches, namely injecting cells expressing the TSH receptor (TSHR) or vaccination with TSHR-DNA in plasmid or adenoviral vectors. These models provide unique insight into several aspects of Graves' disease: 1) manipulating immunity toward Th1 or Th2 cytokines enhances or suppresses hyperthyroidism in different models, perhaps reflecting human disease heterogeneity; 2) the role of TSHR cleavage and A subunit shedding in immunity leading to thyroid-stimulating antibodies (TSAbs); and 3) epitope spreading away from TSAbs and toward TSH-blocking antibodies in association with increased TSHR antibody titers (as in rare hypothyroid patients). Major developments from the models include the isolation of high-affinity monoclonal TSAbs and analysis of antigen presentation, T cells, and immune tolerance to the TSHR. Studies of inbred mouse strains emphasize the contribution of non-MHC vs. MHC genes, as in humans, supporting the relevance of the models to human disease. Moreover, other findings suggest that the development of Graves' disease is affected by environmental factors, including infectious pathogens, regardless of modifications in the Th1/Th2 balance. Finally, developing immunospecific forms of therapy for Graves' disease will require painstaking dissection of immune recognition and responses to the TSHR.

Animals↗
PubMed2005

Human autoantibodies modulate the T cell epitope repertoire but fail to unmask a pathogenic cryptic epitope.

Abs can tune the responses of Ag-specific T cells by influencing the nature of the epitope repertoire displayed by APCs. We explored the interaction between human self-reactive T cells and human monoclonal autoantibodies from combinatorial Ig-gene libraries derived from autoimmune thyroiditis patients and specific for the main autoantigen thyroid peroxidase (TPO). All human mAbs extensively influenced the T cell epitope repertoire recognized by different TPO-specific T cell clones. The action of the human mAbs was complex, because sometimes the same Ab suppressed or enhanced the epitopes recognized by the 10 different TPO-specific T cell clones. The human mAbs could modulate the epitope repertoire when TPO was added exogenously and when expressed constitutively on the surface of APCs. However, they could not unmask an immunodominant cryptic TPO epitope. In this study, we show that human autoantibodies influence the activity of self-reactive T cells and prove their relevance in concealing or exposing epitopes recognized by self-reactive T cells. However, our results further stress the biological significance of the immunodominant cryptic epitope we have defined and its potential importance in the evolution of autoimmunity.

Animals↗
PubMed2005

Gene expression profiles differ markedly in mouse strains that are (or are not) susceptible to hyperthyroidism induced using thyrotropin receptor-expressing adenovirus.

BALB/c mice are susceptible and C57BL/6 mice are resistant to Graves' hyperthyroidism induced by immunization with adenovirus encoding the thyrotropin receptor (TSHR) A-subunit. Both strains develop comparable levels of TSHR antibodies, but potent TSH blocking antibody activity in C57BL/6 mice likely blocks development of hyperthyroidism. We used microarrays to compare gene expression in spleens of mice immunized with A-subunit adenovirus (TSHR-Ad) or control adenovirus (Con-Ad). To preclude the effects of variable thyroxine (T(4)) levels, mice were studied when euthyroid as follows: BALB/c mice immunized three times with TSHR-Ad or Con-Ad and C57BL/6 mice immunized three times with TSHR-Ad or Con-Ad. Among the 14,000 expressed probe sets, there were no statistically significant differences in gene expression in BALB/c mice immunized with TSHR-Ad versus Con-Ad. In contrast, expression of 57 transcripts (representing 40 genes) changed in response to TSHR-Ad in C57BL/6 mice. Diverse genes were identified, including proteins involved in immune responses, inflammation, and cell cycling as well as heat-shock proteins and proteases. Down-regulation of chitinase 3- and-4 gene expression likely reflects cytokines produced by T-helper 2 (Th2) type cells. Indeed, the immunoglobulin (IgG) subclass for TSHR antibodies reflects a deviation away from Th2 cytokines and toward Th1 in C57BL/6 mice. In conclusion, TSHR-Ad immunization altered gene expression profiles in C57BL/6, but not in BALB/c, mice. This response primarily involved reduced gene expression. In C57BL/6 mice, decreased expression of genes such as cathelicidin, calgranulins, and lipocalin following TSHR A-subunit adenovirus immunization suggests the importance of innate immunity in this response.

Adenoviridae↗
PubMed2005

Cathepsin S is not crucial to TSHR processing and presentation in a murine model of Graves' disease.

By regulating invariant (Ii) chain processing and MHC class II peptide loading, the endosomal protease cathepsin S (Cat S) has a potential role in autoimmune susceptibility. Indeed, Cat S null mice are resistant to I-Ab-restricted experimental myasthenia gravis due to inadequate peptide presentation. To explore the role of Cat S in a Graves' disease model, I-Ad-restricted wild-type (WT) and Cat S(-/-) mice were immunized with adenovirus encoding the A subunit of thyroid stimulating hormone receptor (TSHR). TSHR adenovirus immunized mice develop Th1 T cells, TSHR antibodies, and a proportion become overtly hyperthyroid. Although TSHR presentation in vitro was initially impaired in Cat S(-/-) mice, subsequent TSHR presentation in vitro and disease development were similar in both groups but with higher antibody responses in Cat S null mice. WT and Cat S(-/-) mice recognized similar T cell epitopes from a panel of overlapping TSHR peptides. TSHR responses were found to be I-Ad-restricted and Cat S(-/-) I-Ad B cells had marked defects in Ii processing. These data imply that loading of TSHR peptides critical to TSHR antibody responses becomes Ii-independent. Contrasting findings among organ-specific murine autoimmune models imply that potential uses of Cat S inhibitors to ameliorate autoimmunity must be determined empirically.

Adenoviridae↗
PubMed2004

Dissociation between iodide-induced thyroiditis and antibody-mediated hyperthyroidism in NOD.H-2h4 mice.

NOD.H-2h4 mice are genetically predisposed to thyroid autoimmunity and spontaneously develop thyroglobulin autoantibodies (TgAb) and thyroiditis. Iodide administration enhances TgAb levels and the incidence and severity of thyroiditis. Using these mice, we investigated the interactions between TSH receptor (TSHR) antibodies induced by vaccination and spontaneous or iodide-enhanced thyroid autoimmunity (thyroiditis and TgAb). Mice were immunized with adenovirus expressing the TSHR A-subunit (or control adenovirus). Thyroid antibodies, histology, and serum thyroxine levels were compared in animals on a regular diet or on a high-iodide diet (0.05% NaI-supplemented water). Thyroiditis severity and TgAb levels were enhanced by iodide administration and were independent of the type of adenovirus used for immunization. In contrast, TSHR antibodies, measured by TSH-binding inhibition, thyroid-stimulating activity, and TSH-blocking activity, were induced in the majority of animals immunized with TSHR (but not control) adenovirus and were unaffected by dietary iodide. The NOD.2h4 strain of mice was less susceptible than BALB/c or BALB/k mice to TSHR adenovirus-induced hyperthyroidism. Nevertheless, hyperthyroidism developed in approximately one third of TSHR adenovirus-injected NOD.2h4 mice. This hyperthyroidism was suppressed by a high-iodide diet, probably by a nonimmune mechanism. The fact that inducing an immune response to the TSHR had no effect on thyroiditis raises the possibility that the TSHR may not be the target involved in the variable thyroiditis component in some humans with Graves' disease.

Adenoviridae↗
PubMed2004

"Hijacking" the thyrotropin receptor: A chimeric receptor-lysosome associated membrane protein enhances deoxyribonucleic acid vaccination and induces Graves' hyperthyroidism.

Naked DNA vaccination with the TSH receptor (TSHR) does not, in most studies, induce TSHR antibodies and never induces hyperthyroidism in BALB/c mice. Proteins expressed endogenously by vaccination are preferentially presented by major histocompatibility complex class I, but optimal T cell help for antibody production requires lysosomal processing and major histocompatibility complex class II presentation. To divert protein expression to lysosomes, we constructed a plasmid with the TSHR ectodomain spliced between the signal peptide and transmembrane-intracellular region of lysosome-associated membrane protein (LAMP)-1, a lysosome-associated membrane protein. BALB/c mice pretreated with cardiotoxin were primed intramuscularly using this LAMP-TSHR chimera and boosted twice with DNA encoding wild-type TSHR, TSHR A-subunit, or LAMP-TSHR. With each protocol, spleen cells responded to TSHR antigen by secreting interferon-gamma, and 60% or more mice had TSHR antibodies detectable by ELISA. TSH binding inhibitory activity was present in seven, four, and two of 10 mice boosted with TSHR A-subunit, LAMP-TSHR, or wild-type TSHR, respectively. Importantly, six of 30 mice had elevated T4 levels and goiter (5 of 6 with detectable thyroid-stimulating antibodies). Injecting LAMP-TSHR intradermally without cardiotoxin pretreatment induced TSHR antibodies detectable by ELISA but not by TSH binding inhibitory activity, and none became hyperthyroid. These findings are consistent with a role for cardiotoxin-recruited macrophages in which (unlike in fibroblasts) LAMP-TSHR can be expressed intracellularly and on the cell surface. In conclusion, hijacking the TSHR to lysosomes enhances T cell responses and TSHR antibody generation and induces Graves'-like hyperthyroidism in BALB/c mice by intramuscular naked DNA vaccination.

Animals↗
PubMed2004

Graves' hyperthyroidism and the hygiene hypothesis in a mouse model.

Graves' hyperthyroidism is an organ-specific autoimmune disease mediated by stimulatory autoantibodies against the TSH receptor (TSHR; thyroid-stimulating antibodies), causing thyroid hyperplasia and hyperthyroidism. Development of this ailment is well known to be under polygenic and environmental control. For example, we recently demonstrated that parasite helminth Schistosoma mansoni infection suppressed a T helper cell type 1 (Th1)-type anti-TSHR immune response and prevented disease development in our mouse model of Graves' disease using adenovirus coding for the TSHR. In the present study we examined the outcome of infection with Mycobacterium bovis bacillus Calmette-Guerin (BCG), a Th1-promoting infectious pathogen, on Graves' disease. Our results show that prior infection with M. bovis BCG differentiates the TSHR-specific immune response toward a Th1 phenotype, as demonstrated by enhanced secretion of a Th1 cytokine interferon-gamma and impaired production of a Th2 cytokine IL-10 from splenocytes stimulated in vitro with TSHR antigen. M. bovis BCG also significantly suppressed disease induction. These data together with our recent report that coinjection of adenovirus expressing the Th1 cytokine IL-12 induced a Th1-polarized, TSHR-specific immune response without affecting disease development support the hygiene hypothesis, rather than Th1-mediated disease suppression. Thus, some infectious pathogens may influence the development of Graves' disease regardless of their ability to modify the Th1/Th2 balance.

Animals↗
PubMed2004

Schistosoma mansoni and alpha-galactosylceramide: prophylactic effect of Th1 Immune suppression in a mouse model of Graves' hyperthyroidism.

Graves' hyperthyroidism, an organ-specific autoimmune disease mediated by stimulatory thyrotropin receptor (TSHR) autoantibodies, has been considered a Th2-dominant disease. However, recent data with mouse Graves' models are conflicting. For example, we recently demonstrated that injection of BALB/c mice with adenovirus coding the TSHR induced Graves' hyperthyroidism characterized by mixed Th1 and Th2 immune responses against the TSHR, and that transient coexpression of the Th2 cytokine IL-4 by adenovirus skewed Ag-specific immune response toward Th2 and suppressed disease induction. To gain further insight into the relationship between immune polarization and Graves' disease, we evaluated the effect of Th2 immune polarization by helminth Schistosoma mansoni infection and alpha-galactosylceramide (alpha-GalCer), both known to bias the systemic immune response to Th2, on Graves' disease. S. mansoni infection first induced mixed Th1 and Th2 immune responses to soluble worm Ags, followed by a Th2 response to soluble egg Ags. Prior infection with S. mansoni suppressed the Th1-type anti-TSHR immune response, as demonstrated by impaired Ag-specific IFN-gamma secretion of splenocytes and decreased titers of IgG2a subclass anti-TSHR Abs, and also prevented disease development. Similarly, alpha-GalCer suppressed Ag-specific splenocyte secretion of IFN-gamma and prevented disease induction. However, once the anti-TSHR immune response was fully induced, S. mansoni or alpha-GalCer was ineffective in curing disease. These data support the Th1 theory in Graves' disease and indicate that suppression of the Th1-type immune response at the time of Ag priming may be crucial for inhibiting the pathogenic anti-TSHR immune response.

Animals↗
PubMed2004

Directed mutagenesis in region 713-720 of human thyroperoxidase assigns 713KFPED717 residues as being involved in the B domain of the discontinuous immunodominant region recognized by human autoantibodies.

Autoantibodies (aAbs) to thyroid peroxidase (TPO), the hallmark of autoimmune thyroid disease (AITD), recognize conformational epitopes restricted to an immunodominant region (IDR), divided into two overlapping domains A and B. Despite numerous efforts aimed at localizing the IDR and identifying aAb-interacting residues on TPO, only two critical amino acids, Lys(713) and Tyr(772), have been characterized. Precise and complete delineation of the other residues involved in the IDR remains to be defined. By using a recombinant anti-TPO aAb T13, we demonstrated that four regions on TPO are part of the IDR/B; one of them, located between amino acids 713 and 720, is particularly important for the binding of sera from patients suffering from AITD. To precisely define critical residues implicated in the binding of aAb to human TPO, we used directed mutagenesis and expressed the mutants in stably transfected CHO cells. Then we assessed the kinetic parameters involved in the interactions between anti-TPO aAbs and mutants by real-time analysis. We identified (i) the minimal epitope 713-717 recognized by mAb 47 (a reference antibody) and (ii) the amino acids used as contact points for two IDR-specific human monoclonal aAbs TR1.9 (Pro(715) and Asp(717)) and T13 (Lys(713), Phe(714), Pro(715), and Glu(716)). Using a rational strategy to identify complex epitopes on proteins showing a highly convoluted architecture, this study definitively identifies the amino acids Lys(713)-Asp(717) as being the key residues recognized by IDR/B-specific anti-TPO aAbs in AITD.

Animals↗
PubMed2004

Naked deoxyribonucleic acid vaccination induces recognition of diverse thyroid peroxidase T cell epitopes.

Recently, we observed that vaccination of BALB/c mice with thyroid peroxidase (TPO)-DNA in a plasmid is highly effective at inducing antibodies that interact with the immunodominant region recognized by human autoantibodies. We have now analyzed the TPO epitopes recognized by memory T cells in these animals. Splenocytes from TPO-DNA (not control DNA)-vaccinated mice responded to TPO protein antigen, as measured by interferon-gamma production. As a group, TPO-immunized mice recognized 35 of 55 overlapping synthetic peptides that encompass the 814-amino acid TPO ectodomain. In individual mice, between five and 10 peptides induced splenocyte responses. Two T cell epitopes were immunodominant, one of which is also recognized by patients with autoimmune thyroid disease. To explore a potential correlation between T and B cell epitopes, we analyzed serum TPO antibody epitopic fingerprints. No relationship was evident. However, the number of T cell epitopes recognized by individual mice was inversely proportional to recognition of an antibody epitopic subdomain. The diversity of TPO T cell epitopes is in striking contrast to the restricted number of TSH receptor (TSHR) peptides (four of 29) recognized by T cells, as is the paucity of antibodies in the same strain of mice vaccinated with TSHR-DNA. In conclusion, our data highlight differences for both antibody and T cell epitopic recognition in TPO- vs. TSHR-DNA-immunized BALB/c mice. These findings provide insight into mechanisms that may be involved in spontaneous immune responses to two major thyroid autoantigens in humans.

Amino Acid Sequence↗