Role of heme in intracellular trafficking of thyroperoxidase and involvement of H2O2 generated at the apical surface of thyroid cells in autocatalytic covalent heme binding

Thyroperoxidase (TPO) is a glycosylated hemoprotein that plays a key role in thyroid hormone synthesis. We previously showed that in CHO cells expressing human TPO (hTPO) only 2% of synthesized hTPO reaches the cell surface. Herein, we investigated the role of heme moiety insertion in the exit of hTPO from the endoplasmic reticulum. Peroxidase activity at the cell surface and cell surface expression of hTPO were decreased by approximately 30 and approximately 80%, respectively, with succinyl acetone, an inhibitor of heme biosynthesis, and were increased by 20% with holotransferrin and aminolevulinic acid, precursors of heme biosynthesis. Results were similar with holotransferrin plus aminolevulinic acid or hemin, but hemin increased cell surface activity more efficiently (+120%) relative to the control. It had been suggested (DePillis, G., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 8857-8960) that covalent attachment of heme to mammalian peroxidases could be an H2O2-dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H2O2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 microM H2O2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H2O2 was synthesized at the apical cell surface and allowed covalent attachment of heme. Two-day incubation of primocultures of thyroid cells with catalase led to a 30% decrease in TPO activity at the cell surface. In conclusion, we provide compelling evidence for an essential role of 1) heme incorporation in the intracellular trafficking of hTPO and of 2) H2O2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule.     

Generation of a biologically active, secreted form of human thyroid peroxidase by site-directed mutagenesis

Using site-directed mutagenesis, we introduced two stop codons immediately upstream of the putative transmembrane domain in human thyroid peroxidase (hTPO) cDNA, truncating the carboxyl terminus of hTPO (933 amino acids) by 85 residues. Mutated hTPO cDNA, inserted into a eukaryotic expression vector, was stably transfected into Chinese hamster ovary (CHO) cells. Immunoprecipitation of cellular 35S-methionine-labeled proteins with Hashimoto's serum revealed a 105-101 kilodalton doublet. In contrast, cells transfected with wild-type hTPO yielded a 112-105 kilodalton doublet. In pulse-chase experiments, CHO cells expressing the truncated hTPO protein secreted immunoprecipitable TPO into the culture medium after 4 h of chase, with levels accumulating progressively over a 24-h period. In contrast, CHO cells expressing wild-type hTPO released no immunoprecipitable TPO into the culture medium. The secreted, truncated form of hTPO appeared as a single band of lesser electrophoretic mobility, as opposed to the doublet expressed within cells. TPO enzymatic activity was present in conditioned media from CHO cells transfected with the mutated hTPO, but was absent in media from cells expressing wild-type hTPO. The stability of the mutated protein appeared similar to that of wild-type hTPO. In summary, we have generated a mutated, secreted form of hTPO that is enzymatically active and immunologically intact. Our data confirm the existence of a transmembrane domain in hTPO, and that hTPO is predominantly an enzyme with an extracellular orientation. The secreted form of hTPO has the potential for generating large amounts of soluble TPO protein for use in future structural and immunological studies.     

Modelling of Thyroid Peroxidase Reveals Insights into Its Enzyme Function and Autoantigenicity

Thyroid peroxidase (TPO) catalyses the biosynthesis of thyroid hormones and is a major autoantigen in Hashimoto’s disease—the most common organ-specific autoimmune disease. Epitope mapping studies have shown that the autoimmune response to TPO is directed mainly at two surface regions on the molecule: immunodominant regions A and B (IDR-A, and IDR-B). TPO has been a major target for structural studies for over 20 years; however, to date, the structure of TPO remains to be determined. We have used a molecular modelling approach to investigate plausible modes of TPO structure and dimer organisation. Sequence features of the C-terminus are consistent with a coiled-coil dimerization motif that most likely anchors the TPO dimer in the apical membrane of thyroid follicular cells. Two contrasting models of TPO were produced, differing in the orientation and exposure of their active sites relative to the membrane. Both models are equally plausible based upon the known enzymatic function of TPO. The “trans” model places IDR-B on the membrane-facing side of the myeloperoxidase (MPO)-like domain, potentially hindering access of autoantibodies, necessitating considerable conformational change, and perhaps even dissociation of the dimer into monomers. IDR-A spans MPO- and CCP-like domains and is relatively fragmented compared to IDR-B, therefore most likely requiring domain rearrangements in order to coalesce into one compact epitope. Less epitope fragmentation and higher solvent accessibility of the “cis” model favours it slightly over the “trans” model. Here, IDR-B clusters towards the surface of the MPO-like domain facing the thyroid follicular lumen preventing steric hindrance of autoantibodies. However, conformational rearrangements may still be necessary to allow full engagement with autoantibodies, with IDR-B on both models being close to the dimer interface. Taken together, the modelling highlights the need to consider the oligomeric state of TPO, its conformational properties, and its proximity to the membrane, when interpreting epitope-mapping data.     

Localization of the thyroid peroxidase autoantibody immunodominant region to a junctional region containing portions of the domains homologous to complement control protein and myeloperoxidase

Thyroid peroxidase (TPO) autoantibody epitopes are largely restricted to an immunodominant region (IDR) on the extracellular region of the native molecule. Localization of the IDR has been a longstanding and difficult goal. The TPO extracellular region comprises a large myeloperoxidase-like domain, linked to the plasma membrane by two smaller domains with homology to complement control protein (CCP) and epidermal growth factor (EGF), respectively. Recent studies have focused on the CCP- and EGF-like domains as the putative location of the TPO autoantibody IDR. To address this issue, we attempted to express on the surface of transfected cells native TPO in which the CCP- and EGF-like domains were deleted, either together or individually. We used a quartet of human monoclonal autoantibodies that define the TPO IDR, as well as polyclonal TPO autoantibodies in patients' sera, to detect these mutated TPO molecules by flow cytometry. The combined CCP/EGF-like domain deletion did not produce a signal with TPO autoantibodies but did not traffic to the cell surface. In contrast, both monoclonal and polyclonal autoantibodies recognized TPO with the juxtamembrane EGF-like domain deleted equally as well as the wild-type TPO on the cell surface. TPO with the CCP-like domain deleted expressed normally on the cell surface, as determined using the polyclonal mouse antiserum. Nevertheless, this modified TPO molecule was recognized very poorly by both the human monoclonal autoantibodies and the polyclonal autoantibodies in patients' sera. In conclusion, we have clearly excluded the juxtamembrane EGF-like domain as being part of the IDR. In contrast, a component of the CCP-like domain does contribute to the IDR. These data, together with findings from other studies, localize the TPO autoantibody IDR to the junction of the CCP-like domain and the much larger myeloperoxidase-like domain on TPO.     

Autoantibodies to Human Thyroid Peroxidase in Immunoassay and Immunoaffinity Chromatography

A biochemical system of radioimmunoassay of human thyroid peroxidase (TPO) with the use of specific autoantibodies was developed for the first time. This system includes lyophilized preparations of human autoantibodies to TPO, radioiodinated TPO, standards prepared from pure TPO, and the solid-phase protein A as a precipitating agent. An analytical system was used to study the TPO distribution in fractions of thyroid tissue homogenate. Differences in TPO concentrations in mitochondria, microsomes, and supernatant fluid depending on thyroid pathology and tissue storage conditions were found. The behavior of immobilized anti TPO autoantibodies in immunoaffinity chromatography of this microsomal antigen was studied.     

Autoantibodies to human thyroid peroxidase in immunoassay and immunoaffinity chromatography

A biochemical system of radioimmunoassay of human thyroid peroxidase (TPO) with the use of specific autoantibodies was developed for the first time. This system includes lyophilized preparations of human autoantibodies to TPO, radioiodinated TPO, standards prepared from pure TPO, and the solid-phase protein A as a precipitating agent. An analytical system was used to study the TPO distribution in fractions of thyroid tissue homogenate. Differences in TPO concentrations in mitochondria, microsomes, and supernatant fluid depending on thyroid pathology and tissue storage conditions were found. The behavior of immobilized anti-TPO-autoantibodies in immunoaffinity chromatography of this microsomal antigen was studied.     

Localization of the discontinuous immunodominant region recognized by human anti-thyroperoxidase autoantibodies in autoimmune thyroid diseases

The discontinuous immunodominant region (IDR) recognized by autoantibodies directed against the thyroperoxidase (TPO) molecule, a major autoantigen in autoimmune thyroid diseases, has not yet been completely localized. By using peptide phage-displayed technology, we identified three critical motifs, LXPEXD, QSYP, and EX(E/D)PPV, within selected mimotopes which interacted with the human recombinant anti-TPO autoantibody (aAb) T13, derived from an antibody phage-displayed library obtained from thyroid-infiltrating TPO-selected B cells of Graves' disease patients. Mimotope sequence alignment on the TPO molecule, together with the binding analysis of the T13 aAb on TPO mutants expressed by Chinese hamster ovary cells, demonstrated that regions 353-363, 377-386, and 713-720 from the myeloperoxidase-like domain and region 766-775 from the complement control protein-like domain are a part of the IDR recognized by the recombinant aAb T13. Furthermore, we demonstrated that these regions were involved in the binding to TPO of sera containing TPO-specific autoantibodies from patients suffering from Hashimoto's and Graves' autoimmune diseases. Identification of the IDR could lead to improved diagnosis of thyroid autoimmune diseases by engineering "mini-TPO" as a target autoantigen or designing therapeutic peptides able to block undesired autoimmune responses.     

Thyroperoxidase, an auto-antigen with a mosaic structure made of nuclear and mitochondrial gene modules.

A lambda gt11 cDNA library was constructed from a normal human thyroid and screened with a rabbit anti-porcine thyroperoxidase antibody. A series of thyroperoxidase (TPO) clones were obtained which allowed determination of the complete primary structure of the protein. The library was also screened with serum from a patient with Hashimoto's thyroiditis, an autoimmune disease characterized by the presence in the serum of high titers of autoantibodies directed against the 'microsomal antigen' (McAg). Comparison of the cDNA sequences from TPO clones and McAg clones provides definite proof that the McAg is TPO. A short segment of TPO was characterized as bearing a major epitope involved in autoimmunity. The primary structure of TPO was 42% homologous to myeloperoxidase (MPO). It contains, in addition, a C-terminal extension with a membrane anchor region contiguous to two domains encoded by modules belonging to the EGF and C4b gene families. The existence in TPO of still another domain presenting a significant homology with a putative heme-binding region of cytochrome C oxidase polypeptide I raises the possibility that a mitochondrial gene module has contributed a piece to the evolution of a typical nuclear mosaic gene.     

Tissue polarity genes of Drosophila regulate the subcellular location for prehair initiation in pupal wing cells

The purpose of this study was to explore the functional role of the cytoplasmic domain of the alpha subunit of the alpha 5/beta 1 integrin, a fibronectin receptor. Mutant CHO cells that express very low levels of endogenous hamster alpha 5 subunit (CHO clone B2) were transfected with an expression vector containing full-length or truncated human alpha 5 cDNAs to form chimeric human alpha 5/hamster beta 1 integrins. Three transfectants were examined: B2a27 expresses a full-length human alpha 5 subunit with 27 amino acids in the cytoplasmic domain; B2a10 expresses an alpha 5 with a 17-amino acid cytoplasmic truncation; B2a1 expresses an alpha 5 with a 26-amino acid truncation. Levels of alpha 5/beta 1 surface expression in B2a27 and B2a10 cells were similar to that in wild type CHO cells. The expression of alpha 5/beta 1 in B2a1 cells was less, amounting to 15-20% of WT levels, despite message levels that were three to five times greater than those of B2a27. The transfectants were used to examine the role of the alpha 5 cytoplasmic domain in cell adhesion, cell motility, cytoskeletal organization, and integrin-mediated tyrosine phosphorylation. The adhesion characteristics of B2a27 and B2a10 cells on fibronectin substrata were similar to each other and to wild type CHO cells. B2a1 cells displayed slight reductions in the strength and rate of adhesion to fibronectin. Cell motility in the presence of fibronectin was similar for B2a27, B2a10, and wild type CHO cells, while the B2a1 cells were substantially less motile. Comparable degrees of cell spreading and extensive organization of actin filaments were observed for B2a27, B2a10, and wild type CHO cells on fibronectin substrata. The B2a1 cells spread to a lesser degree, and some organization of actin was observed; the untransfected B2 cells remained round on fibronectin substrata and showed no actin reorganization. Since the reduced motility and cell spreading observed in the B2a1 cells might be due either to reduced surface expression of alpha 5/beta 1 or to the truncation in the alpha 5 cytoplasmic domain, we used flow cytometric cell sorting to select populations of B2a1 and B2a27 cells expressing similar levels of cell surface alpha 5. The deficits in spreading and motility were present in B2a1 cells expressing high levels of alpha 5. Thus the region of the alpha 5 cytoplasmic domain adjacent to the membrane seems to play an important role in cytoskeletal organization and cell motility. We also examined whether alpha subunit truncation would affect integrin- mediated tyrosine phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Elevated blood Hsp60, its structural similarities and cross-reactivity with thyroid molecules,and its presence on the plasma membrane of oncocytes point to the chaperonin as an immunopathogenic factor in Hashimoto's thyroiditis

The role Hsp60 might play in various inflammatory and autoimmune diseases is under investigation, but little information exists pertaining to Hashimoto’s thyroiditis (HT). With the aim to fill this gap, in the present work, we directed our attention to Hsp60 participation in HT pathogenesis. We found Hsp60 levels increased in the blood of HT patients compared to controls. The chaperonin was immunolocalized in thyroid tissue specimens from patients with HT, both in thyrocytes and oncocytes (Hurthle cells) with higher levels compared to controls (goiter). In oncocytes, we found Hsp60 not only in the cytoplasm but also on the plasma membrane, as shown by double immunofluorescence performed on fine needle aspiration cytology. By bioinformatics, we found regions in the Hsp60 molecule with remarkable structural similarity with the thyroglobulin (TG) and thyroid peroxidase (TPO) molecules, which supports the notion that autoantibodies against TG and TPO are likely to recognize Hsp60 on the plasma membrane of oncocytes. This was also supported by data obtained by ELISA, showing that anti-TG and anti-TPO antibodies cross-react with human recombinant Hsp60. Antibody-antigen (Hsp60) reaction on the cell surface could very well mediate thyroid cell damage and destruction, perpetuating inflammation. Experiments with recombinant Hsp60 did not show stimulation of cytokine production by peripheral blood mononuclear cells from HT patients. All together, these results led us to hypothesize that Hsp60 may be an active player in HT pathogenesis via an antibody-mediated immune mechanism.     

Protein radical formation on thyroid peroxidase during turnover as detected by immuno-spin trapping

Thyroid peroxidase (TPO) is a 933 amino acid residue, heme-containing, integral membrane glycoprotein that catalyzes two steps in the maturation of the thyroid hormone precursor. As with other peroxidases, these reactions require hydrogen peroxide and initial enzyme oxidation. Previous researchers studied the oxidative state of the TPO heme moiety using spectrophotometric and catalytic analyses. We use a novel antiserum to 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to detect radical-derived DMPO spin-trapped TPO. Our work reveals that TPO generates radical adducts in the presence of H2O2, but that the generation of these adducts can be suppressed by the addition of substrates and inhibitors. Chemical alteration of the tyrosine residues of TPO greatly reduces the generation of TPO-DMPO adducts. Iodide strongly suppresses the H2O2-generated production of TPO radical adducts and protects the enzyme from loss of enzyme activity. Because the normal catalytic mechanism of TPO involves the production of radical species, TPO is potentially more susceptible to oxidative damage than most enzymes which do not require H2O2 as a substrate. We hypothesize that oxidatively damaged TPO may trigger the production of anti-TPO autoantibodies, resulting in the development of autoimmune thyroid disorders. Evidence that correlates iodine deficiencies with development of thyroid autoimmune disorders supports this conjecture.     

Analysis of transport and targeting of syndecan-1: effect of cytoplasmic tail deletions.

Madin-Darby canine kidney (MDCK) cells and Chinese hamster ovary (CHO) cells were transfected with wild-type and cytoplasmic deletion mutants of mouse syndecan-1 to study the requirements for transport and polarized expression of this proteoglycan. Expression in MDCK cells revealed that wild-type syndecan-1 is directed to the basolateral surface via a brefeldin A-insensitive route. A deletion of the last 12 amino acids of the syndecan-1 cytoplasmic tail (CT22) was sufficient to result in the appearance of mutant proteoglycans at both the basolateral and apical cell surfaces. Treatment with brefeldin A was able to prevent apical transport of the mutants. We thus propose that the C-terminal part of the cytoplasmic tail is required for steady-state basolateral distribution of syndecan-1. In CHO cells a deletion of the last 25 or 33 amino acids of the 34-residue cytoplasmic domain (CT9 and CT1, respectively) resulted in partial retention of the mutants in the endoplasmic reticulum (ER). A deletion mutant lacking the last 12 amino acids (CT22) was not retained. Interestingly, the unglycosylated core proteins of the CT9 and CT1 mutants showed a significantly lower apparent molecular weight when analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis than wild-type syndecan-1. However, when CHO transfectants expressing the CT1 mutant were incubated with brefeldin A, causing fusion of the ER and Golgi, CT1 ran with an almost equally high apparent molecular weight as the wild-type molecule. This would suggest that syndecan-1 undergoes extensive posttranslational modifications or forms an SDS-resistant dimer/complex after transit from the ER.     

Effects of inorganic iodide, epidermal growth factor and phorbol ester on hormone synthesis by porcine thyroid follicles cultured in suspension.

Porcine thyroid follicles cultured in suspension for 96 h synthesized and secreted thyroid hormones in the presence of thyrotropin (TSH). The secretion of newly synthesized hormones was assessed by determining the contents of thyroxine (T4) and triiodothyronine (T3) in the media and by paperchromatographic analysis of 125I-labelled hormones in the media where the follicles were cultured in the presence and absence of inhibitors of hormone synthesis. The hormone synthesis and secretion was modified by exogenously added NaI (0.1-100 microM). The maximal response was obtained at 1 microM. Thyroid peroxidase (TPO) activity in the cultured follicles with TSH for 96 h was dose-dependently inhibited by NaI. One hundred microM of NaI completely inhibited TSH-induced TPO activity. Moreover, both epidermal growth factor (EGF: 10(-9) and 10(-8) M) and phorbol 12-myristate 13-acetate (PMA: 10(-8) and 10(-7) M) inhibited de novo hormone synthesis. An induction of TPO activity by TSH was also inhibited by either agent. These data provide direct evidences that thyroid hormone synthesis is regulated by NaI as well as TSH at least in part via regulation of TPO activity and also that both EGF and PMA are inhibitory on thyroid hormone formation.     

A human Fab fragment specific for thyroid peroxidase generated by cloning thyroid lymphocyte-derived immunoglobulin genes in a bacteriophage lambda library.

A human Fab fragment (SP2) which binds specifically to human thyroid peroxidase has been generated by expressing random combinations of heavy and light chain immunoglobulin genes (derived from Graves' thyroid cDNA) in a bacteriophage lambda library. In common with many serum TPO autoantibodies, the cloned Fab fragment is IgG1 kappa and has a high affinity for TPO (approximately 10(-9) M). On the basis of their nucleotide sequences, the heavy and light chain genes coding for SP2 belong to families VHI, (D), JH3 and VKI, JK2, respectively. These data provide the first characterization at a molecular level of a human thyroid peroxidase antibody associated with autoimmune thyroid disease.     

Thyroid autoantigens and their relevance in the pathogenesis of thyroid autoimmunity

Humoral and cellular immune responses are both involved in autoimmune disorders of the thyroid gland. In the last five years, new substantial data have been obtained on the nature and the expression of thyroid cell surface autoantigens and on the demonstration of the functional heterogeneity of autoantibodies to the thyroid stimulating hormone (TSH) receptor. In the present report, attention will be mainly focused on recent studies carried out in our laboratory. The main autoantigens so far identified include the 'microsomal' antigen, thyroglobulin and the TSH receptor. For many years the 'microsomal' antigen (M) was considered a poorly characterized constituent of the cytoplasm of the thyroid cell. In the last five years, several lines of evidence were provided indicating that M is also well represented on the surface of the follicular cell and is identical to thyroid peroxidase (TPO). The use of anti-TPO monoclonal antibodies, presently available, have confirmed this antigenic identity. Microsomal (anti-TPO) antibodies are very useful markers of autoimmune thyroid disorders and are generally present in Hashimoto's thyroiditis, idiopathic myxedema and Graves' disease. TSH receptor antibodies (TRAb) are present in the sera of patients with Graves' disease. TRAb are able to stimulate thyroid adenylate cyclase and also to mimic TSH in its thyroid growth stimulation. Thus, these antibodies may have a pathogenetic role in goiter formation and in thyroid hyperfunction in Graves' disease. TRAb were also shown to inhibit both TSH binding to its receptor and TSH-stimulated adenylate cyclase activity. Recently TRAb, which inhibited TSH-stimulated adenylate cyclase activity, were found in idiopathic myxedema patients and may be responsible for impairment of thyroid function.(ABSTRACT TRUNCATED AT 250 WORDS)     

An alternative form of the integrin beta 1 subunit with a variant cytoplasmic domain.

We have identified an alternatively spliced form of the human beta 1 integrin subunit, beta 1S. The beta 1S mRNA is expressed in human platelets, HEL and K562 erythroleukemia cell lines, and THP1 monocytic and HL60 promyelocytic cell lines. It is undetectable in peripheral blood lymphocytes and cultured umbilical vein endothelial cells at early passages. The beta 1S cDNA encodes a new cytoplasmic domain distinct from the previously reported alternative cytoplasmic domain of the beta 1 subunit. The sequence reveals the presence of an insert of 116 nucleotides which produces a frame shift in the previously reported 3' end of the beta 1 integrin subunit and codes for a unique 48-amino acid COOH-terminal sequence. An antiserum prepared against a synthetic peptide generated from the deduced sequence of the beta 1S cytoplasmic domain immunoprecipitated an HEL cell surface molecule that comigrated with the usual beta 1 subunit in sodium dodecyl sulfate electrophoresis. The immunoprecipitation indicated that beta 1S constitutes a minor portion of total beta 1 subunit in these cells. This variant beta 1 cytoplasmic tail may modulate integrin affinity and may provide additional modes for the transduction of extracellular signals and modulation of cytoskeletal organization by beta 1 integrins.     

Immunoglobulin G subclasses of anti-thyroid peroxidase autoantibodies in human autoimmune thyroid diseases

A distribution of immunoglobulin G (IgG) subclass of anti-thyroid peroxidase (TPO) autoantibodies was studied to know whether anti-TPO autoantibodies are closely implicated in the pathogenesis of human autoimmune thyroid diseases. As a result of analyzing 14 patients' sera, 7 with Graves' disease and 7 with Hashimoto's thyroiditis, anti-TPO autoantibodies were found to consist of mainly IgG1 subclass. Percentages of both IgG1 and IgG2 subclasses in IgG class of autoantibodies corresponded to those in the normal serum composition, whereas IgG3 subclass was scarcely contained in anti-TPO autoantibodies and IgG4 subclass markedly increased. It was thought that anti-TPO autoantibodies had a capability to lyse thyroid follicular cells by the mechanism of antibody-dependent complement-mediated cytolysis, because IgG1 and IgG2 subclasses of antibodies can fix complement and TPO locates in apical membrane surface of thyroid follicular cells. Comparing Graves' disease with Hashimoto's thyroiditis, mean percentages of both IgG1 and IgG2 subclasses of 2 groups were statistically different. Namely, sera of patients with Graves' disease had higher and lower mean percentages of IgG1 and IgG2 subclasses of autoantibodies, respectively, than those with Hashimoto's thyroiditis, though no plausible explanation for these differences can be offered at the present time.     

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.