Polyreactive Autoantibodies Research Articles

Autoimmune B Cell Repertoire in a Mouse Model of Sjögren's Syndrome.

In genetically prone individuals, chronic immune activation may lead to expansion of autoreactive lymphocyte clones that can induce organ damage developing autoimmune disorders. Sjögren’s Syndrome (SjS) is a systemic chronic autoimmune disease that primarily affects exocrine glands. Despite the accumulated evidences of profound B-cell alterations of humoral immunity, the repertoire and development of B-cell autoreactivity in SjS remains to be determined. We hypothesize that SjS mice will have an increased frequency of self-reactive B cells with a progressive evolution to antigen-driven oligoclonality. Here, we study the B cell repertoire of NOD.H-2h4 mice, a mouse model of spontaneous autoimmunity mimicking SjS without developing diabetes. A library of 168 hybridomas from NOD.H-2h4 mice and 186 C57BL/6J splenocytes at different ages was created. The presence of mono or polyreactive autoantibodies to several antigens was evaluated by ELISA, and their staining patterns and cellular reactivity were tested by IFA and FACS. We observed a higher frequency of autoreactivity among B-cell clones from NOD.H-2h4 mice as compared to wild-type mice. The presence of polyreactive and autoreactive IgG clones increased with mice age. Strikingly, all anti-Ro52 autoantibodies were polyreactive. No loss of polyreactivity was observed upon antibody class switching to IgG. There was a progression to oligoclonality in IgG B cells with mice aging. Our results indicate that in the NOD.H-2h4 mouse model of SjS, IgG+ B cells are mainly polyreactive and might expand following an unknown antigen-driven positive selection process.

Expansion of polyreactive and autoreactive IgG B cells in NOD.H-2h4 mice, a model of Sjögren’s Syndrome

Abstract In genetically prone individuals, chronic immune activation may lead to expansion of autoreactive lymphocyte clones that can induce organ damage developing autoimmune disorders. Sjögren’s Syndrome (SjS) is one of the most common systemic chronic autoimmune diseases that primarily affects exocrine glands with progressive dryness of mouth and eyes. Despite the accumulated evidences of profound B-cell alterations of humoral immunity, the repertoire and onset of B-cell autoreactivity in SjS remains to be determined. We hypothesize that SjS mice will have an increased frequency of self-reactive B cells with a progressive evolution to antigen-driven oligoclonality. Here, we study the B cell repertoire of NOD.H-2h4 mice, a mouse model of spontaneous autoimmunity mimicking SjS without developing diabetes. All female mice presented salivary gland infiltrates and autoantibodies such as anti-dsDNA and anti-Ro52, both characteristic of SjS, at the age of 24 weeks. A library of 456 hybridomas from NOD.H-2h4 mice splenocytes at three different ages: 28, 47 and 66 weeks old was created. The presence of mono or polyreactive autoantibodies against dsDNA, Ro52, LPS, insulin, histones and OVA was evaluated by ELISA, and their staining patterns and cellular reactivity were tested by IFA and FACS. We observed a high percentage of autoreactivity among B-cell clones from NOD.H-2h4 mice. IgM autoreactive B cells were maintained, while polyreactive and autoreactive IgG clones increased with mice age. Interestingly, all anti-Ro52 autoantibodies were polyreactive. Our results indicate that in the NOD.H-2h4 mouse model of SjS, IgG+ B cells are mainly polyreactive and might expand following an unknown antigen-driven positive selection process.

Computer-aided Drug Designing for the Identification of High-Affinity Small Molecule Targeting CD20 for the Clinical Treatment of Chronic Lymphocytic Leukemia (CLL).

Chronic lymphocytic leukemia (CLL) is a B-lineage lymphoid malignancy of self-reactive cells that are focused to produce polyreactive natural autoantibodies. Its surface protein marker CD20 plays an important role in the humoral immune response targeting which has emerged as an attractive therapeutic option for the treatment of CLL. The present study explains the interaction of the CD20 with its established inhibitors and to discover the compound having high binding affinity against the target protein receptor. Technically, during the development of new compound through docking studies, best drug among all pre-exist drugs got filtered, hence in reference to docked best drug study moved ahead. The 3D structure of CD20 was built using homology base fold recognition method using Smith waterman's Local alignment and standalone Delta Blast algorithms. 23 established inhibitors towards CD20 were selected in this present investigation. Among these inhibitors, etoposide (RMSD value -96.6481) showed high binding capacity with the receptor CD20 which was further subjected to virtual screening. The said screening presented 380 possible drugs having structural similarity to etoposide. The docking studies of the screened drugs separated the compound having PubChem CID: 11753896 (RMSD value -98.5416). Toxicity and interaction profile validated this compound for having a better affinity with the target protein. Conclusively, this research study says that according to ADMET profile and BOILED-Egg plot, the compound (PubChem CID: 11753896) obtained from Virtual Screen could be the best drug in future during the prevention of Chronic Lymphocytic Leukemia. The compound identified in the present investigation can be subjected further for in vitro and in vivo studies for ADMET properties and it could optimize a good profile in the field of pharmacy and bioavailable for suppressing cancer. The pharmacophore study revealed that the drug CID11753896 is a non-inhibitor of CYP450 microsomal enzymes and was found to be non-toxic, similar to the established compound CID36462. It has a lower LD50 value of 2.5423mol/kg as compared to the established compound whose LD50 value is 2.9588mol/kg. Also, the compound was found to be non-carcinogenic.

Naïve B cells are prone to develop into polyreactive autoantibody secreting cells from adult RAG2-deficient patient with combined immunodeficiency

Naïve B cells are prone to develop into polyreactive autoantibody secreting cells from adult RAG2-deficient patient with combined immunodeficiency

Associations of Autoimmunity, Immunodeficiency, Lymphomagenesis, and Gut Microbiota in Mice with Knockins for a Pathogenic Autoantibody

A number of mouse strains transgenic for B-cell receptors specific for nucleic acids or other autoantigens have been generated to understand how autoreactive B cells are regulated in normal and autoimmune mice. Previous studies of nonautoimmune C57BL/6 mice heterozygous for both the IgH and IgL knockins of the polyreactive autoantibody, 564, produced high levels of autoantibodies in a largely Toll-like receptor 7-dependent manner. Herein, we describe studies of mice homozygous for the knockins that also expressed high levels of autoantibodies but, unlike the heterozygotes, exhibited a high incidence of mature B-cell lymphomas and enhanced susceptibility to bacterial infections. Microarray analyses and serological studies suggested that lymphomagenesis might be related to chronic B-cell activation promoted by IL-21. Strikingly, mice treated continuously with antibiotic-supplemented water did not develop lymphomas or abscesses and exhibited less autoimmunity. This mouse model may help us understand the reasons for enhanced susceptibility to lymphoma development exhibited by humans with a variety of autoimmune diseases, such as Sjögren syndrome, systemic lupus erythematosus, and highly active rheumatoid arthritis.

Role of Natural Autoantibodies and Natural IgM Anti-Leucocyte Autoantibodies in Health and Disease.

We review how polyreactive natural IgM autoantibodies (IgM-NAA) protect the host from invading micro-organisms and host neo-antigens that are constantly being produced by oxidation mechanisms and cell apoptosis. Second, we discuss how IgM-NAA and IgM anti-leukocyte antibodies (IgM-ALA) inhibits autoimmune inflammation by anti-idiotypic mechanisms, enhancing removal of apoptotic cells, masking neo-antigens, and regulating the function of dendritic cells (DC) and effector cells. Third, we review how natural IgM prevents autoimmune disorders arising from pathogenic IgG autoantibodies, triggered by genetic mechanisms (e.g., SLE) or micro-organisms, as well as by autoreactive B and T cells that have escaped tolerance mechanisms. Studies in IgM knockout mice have clearly demonstrated that regulatory B and T cells require IgM to effectively regulate inflammation mediated by innate, adaptive, and autoimmune mechanisms. It is, therefore, not surprising why the host positively selects such autoreactive B1 cells that generate IgM-NAA, which are also evolutionarily conserved. Fourth, we show that IgM-ALA levels and their repertoire can vary in normal humans and disease states and this variation may partly explain the observed differences in the inflammatory response after infection, ischemic injury, or after a transplant. We also show how protective IgM-NAA can be rendered pathogenic under non-physiological conditions. We also review IgG-NAA that are more abundant than IgM-NAA in plasma. However, we need to understand if the (Fab)2 region of IgG-NAA has physiological relevance in non-disease states, as in plasma, their functional activity is blocked by IgM-NAA having anti-idiotypic activity. Some IgG-NAA are produced by B2 cells that have escaped tolerance mechanisms and we show how such pathogenic IgG-NAA are regulated to prevent autoimmune disease. The Fc region of IgG-NAA can influence inflammation and B cell function in vivo by binding to activating and inhibitory FcγR. IgM-NAA has therapeutic potential. Polyclonal IgM infusions can be used to abrogate on-going inflammation. Additionally, inflammation arising after ischemic kidney injury, e.g., during high-risk elective cardiac surgery or after allograft transplantation, can be prevented by pre-emptively infusing polyclonal IgM or DC pretreated ex vivo with IgM or by increasing in vivo IgM with a vaccine approach. Cell therapy is appealing as less IgM will be required.

Fatty Acid Amide Hydrolase Regulates Peripheral B Cell Receptor Revision, Polyreactivity, and B1 Cells in Lupus.

C57BL/6 mice bearing the Sle2(z) lupus-susceptibility congenic interval on chromosome 4 display high titers of polyclonal autoantibodies with generalized B cell hyperactivity, hallmarks of systemic lupus erythematosus. In B6.Sle2(z)HEL(Ig).sHEL BCR-transgenic mice, Sle2(z) did not breach central tolerance, but it led to heightened expression of endogenous Ig H and L chains in splenic B cells, upregulation of RAG, and serological polyreactivity, suggestive of excessive receptor revision. Fatty acid amide hydrolase (FAAH), a gene in the minimal subcongenic interval generated through recombinant mapping, was found to be upregulated in Sle2(z) B cells by microarray analysis, Western blot, and functional assays. Pharmacological inhibition of FAAH reversed the increase in receptor revision, RAG expression, and polyreactive autoantibodies in lupus-prone mice. These studies indicate that increased peripheral BCR revision, or selective peripheral expansion of BCR-revised B cells, may lead to systemic autoimmunity and that FAAH is a lupus-susceptibility gene that might regulate this process.

Aberrant levels of natural IgM antibodies in osteoarthritis and rheumatoid arthritis patients in comparison to healthy controls

Natural IgM antibodies (nIgM) are polyreactive autoantibodies that have diverse roles in regulating autoimmunity, systemic inflammation and removal of oxidized low-density lipoproteins (oxLDL). We hypothesized that aberrant states of nIgM may exist in persons with osteoarthritis (OA) and rheumatoid arthritis (RA). Herein, we characterized and compared the levels of nIgM specific for phosphorylcholine (anti-PC), double-stranded DNA (anti-dsDNA), and galactosyl (anti-Gal) in persons with OA, RA and healthy controls (HC). Levels of anti-PC nIgM in OA patients were significantly lower than both HC and RA patients in an age-adjusted analysis (P<0.05). In contrast, anti-Gal nIgM levels were significantly higher in RA patients than OA patients (P<0.05) and markedly increased in comparison to HC. Anti-PC nIgM significantly correlated with anti-dsDNA and anti-Gal nIgM levels in HC and RA (P<0.05) but not in OA patients. Elevated CRP levels were associated with RA conditions and old ages in general. There was no significant correlation between anti-PC nIgM and CRP or oxLDL levels. Our study highlights for the first time the evidence of aberrant state of nIgM in human OA compared to healthy individuals that implicates a deficiency in immune responses to oxLDL which may contribute to the metabolic syndromes in the development of OA.

Polyreactive Monoclonal Autoantibodies in Multiple Sclerosis: Functional Selection from Phage Display Library and Characterization by Deep Sequencing Analysis

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system that primarily affects young and middle-aged people. It is widely accepted that B lymphocyte activation is required for MS progression. Despite the fact that the exact triggering mechanisms of MS remain enigmatic, one may suggest that MS can be induced by viral or bacterial infection in combination with specific genetic and environmental factors. Using deep sequencing and functional selection methodologies we characterized clones of poly- and cross-reactive antibodies that are capable of simultaneous recognition of viral proteins and autoantigens. The latter, in turn, possibly may trigger MS progression through molecular mimicry. It was identified that two cross-reactive antigens are probably recognized by light or heavy chains individually. According to the high structural homology between selected autoantibodies and a number of various antiviral IgGs, we suggest that a wide range of pathogens, instead of a single virus, be regarded as possible triggers of MS.

Polyreactive Monoclonal Autoantibodies in Multiple Sclerosis: Functional Selection from Phage Display Library and Characterization by Deep Sequencing Analysis

Polyreactive Monoclonal Autoantibodies in Multiple Sclerosis: Functional Selection from Phage Display Library and Characterization by Deep Sequencing Analysis

Autoantibodies—epiphenomena or biological clues

The potential roles for autoantibodies in renal transplantation are increasing, as illustrated by a recent report of polyreactive autoantibodies produced by B-cell clones from a kidney transplant recipient that can bind apoptotic cells and activate complement. Such autoantibodies have the potential to amplify microcirculation injury caused by alloantibody in antibody-mediated transplant rejection.

Colostrum of Healthy Mothers Contains Broad Spectrum of Secretory IgA Autoantibodies

Human colostrum and milk provide a newborn with immunomodulatory components, ensuring protection and proper development of the immune system. Secretory IgA antibodies in colostrum represent the first line of defence against harmful substances, but their potential spectra of reactivity with autoantigens remains unclear. Here, we characterised the repertoire of natural sectretory IgA autoantibodies in colostrum of healthy mothers. The human colostrum samples from 39 healthy mothers were analyzed for autoantibodies by indirect immunofluorescence, dot blots, immunoblots and ELISA. We found that there is high diversity in reactivities of colostral IgA antibodies to autoantigens among individual samples. Using tissue sections and biochips commonly used for autoimmunity testing, we found that most samples reacted with monkey ovary (79.3%), monkey pancreatic tissue (78.6%), human HEp-2 cells (69%) and monkey adrenal gland (69.0%), fewer samples reacted with monkey liver tissue (47.2%), rat stomach (42.9%), monkey testicular tissue (41.4%), monkey salivary gland (39.3%), rat kidney (32.1%) and monkey cerebellar tissue (17.9%). At the protein level, we detected reactivity of IgA with 21 out of 25 (auto) antigens. The majority of the samples reacted with the pyruvate dehydrogenase complex, E3 ubiquitin ligase, cytosolic liver antigen, promyelocytic leukemia protein and nuclear pore glycoprotein-210. Using ELISA, we found reactivity of colostral IgA antibodies against examined extractable nuclear antigens, double stranded DNA, phospholipids and neutrophil cytoplasm. The broad spectrum of polyreactive natural autoantibodies present in human colostrum may contribute to proper development of mucosal immune system of the breastfed infant.

The relevance of antigen binding to the pathogenicity of lupus autoantibodies

Although lupus autoantibodies provide diagnostic value, discordance between serum levels and nephritis poses mechanistic questions. Krishnan and co-workers report that only those that crossreact with basement membrane components produce immune deposits. Thus, other glomerular binding properties probably define where deposits form. Thereafter, Fc- and complement-mediated events influence disease expression. Clearly other factors determine the ultimate phenotype; however, the findings provide insights into the variable disease patterns in lupus nephritis.

Murine lupus strains differentially model unique facets of human lupus serology

Systemic lupus erythematosus (SLE) is a polygenic autoimmune disease characterized by the production of anti-nuclear autoantibodies that lead to subsequent end organ damage. Previous array-based studies in patients with SLE have shown that high immunoglobulin (Ig)G anti-nuclear autoantibody reactivity was associated with severe renal lupus, whereas IgM polyreactivity was associated with less severe disease. To ascertain how different murine lupus strains recapitulate these different autoantibody profiles seen in patients, serum from New Zealand black (NZB)/NZ white (W) F(1), Murphy Roths large (MRL)/lpr, NZ mixed (M)2410 and BXSB strains were compared using a comprehensive array-based screen. The array results were verified using enzyme-linked immunosorbent assays (ELISAs). Serum from MRL/lpr mice exhibited high levels of IgG anti-nuclear antibodies as well as anti-glomerular antibodies and variable levels of antibodies to myosin, Matrigel and thyroglobulin. Elevated anti-nuclear IgG antibodies were associated with severe nephritis in this strain. In contrast, NZM2410 mice exhibited lower IgG autoantibody levels with less severe nephritis but a significantly higher polyreactive IgM autoantibody profile. ELISA analysis confirmed these results. The NZB/NZW F(1) and BXSB strains exhibited an intermediate serological profile. Hence, just as in patients with SLE, whereas strong IgG reactivity to nuclear antigens is associated with severe renal disease, a polyreactive IgM seroprofile is also less ominous in murine lupus.

A matter of debate in chronic lymphocytic leukemia

Autoimmune cytopenia is a noninfrequent complication of chronic lymphocytic leukemia (CLL) classified into simple and complex autoimmunity, on the basis of the absence or the concomitance of disease progression. Simple-refractory and complex autoimmunity indicate the need of treatment. Here, we review the epidemiology and the biological mechanisms that account for CLL-associated autoimmune disorders and discuss their prognostic relevance and the indication of treatment. Autoimmune disorders can complicate CLL at any stage and even occur in the preleukemic monoclonal B lymphocytosis. CLL cells can act as antigen-presenting cells, possibly inducing the formation of autoreactive T helper cells (through the production of B-cell activator factor and a proliferation-inducing ligand) and nonfunctional T regulatory cells (via CD27-CD70 interaction). Further, nonmalignant lymphocytes may stimulate via CD154-mediated mechanism both tumor growth and the development of autoimmunity, especially after fludarabine-based regimens of therapy. CLL cells tend to produce monoclonal polyreactive autoantibodies suggesting that autoantigen stimulation via B-cell receptor signaling may affect the natural history of CLL. Though the results of multivariate analysis do not allow to separate autoimmune disorders developing in CLL from conventional prognostic factors, the pathogenetic intertwining between autoimmunity and malignant transformation indicates the importance of defining whether the occurrence of autoimmunity in CLL might be considered an autonomous prognostic indicator that influences treatment decisions. Simple-refractory and complex autoimmunity are independent indicators of therapy for CLL. Further, epidemiological and biological studies will help clarifying the prognostic and possibly also the pathogenetic significance of simple autoimmunity.

Contribution of VH replacement products to the generation of autoantibodies in systemic lupus erythematosus (44.19)

Abstract Systemic lupus erythematosus (SLE) is characterized by the over production of high affinity autoantibodies that play important roles in SLE pathogenesis. However, it is not clear how these antibodies are generated and selected during the course of lupus. Our sequence analysis show that VH replacement products are highly enriched in IgH genes derived from different autoimmune diseases, including SLE. To gain insight into the mechanisms responsible for autoantibody generation in SLE, we performed single cell PCR analysis to study the functional antibody repertoires in active SLE patients. Analysis of 102 recombinant antibodies derived from the naive B cells and plasmablasts of the SLE patients show that 4.8% of the antibodies derived from naïve B cells and 26.7% of the antibodies derived from plasmablasts are autoreactive. Detailed sequence analysis reveal that in the plasmablasts of two SLE patients, VH replacement products contribute to more than 30% of IgH genes. These VH replacement products have long CDR3 regions enriched with charged amino acids, and more than 60% of them encode anti-nuclear or poly-reactive autoantibodies. These studies provide the first evidence that VH replacement products directly contribute to the generation of autoantibodies in SLE.

Polyreactive autoantibodies in systemic lupus erythematosus have pathogenic potential

The present study was undertaken to determine whether germline encoded and polyreactive antibodies might be pathogenic and whether the breach of early tolerance checkpoints in systemic lupus erythematosus (SLE) might lead to a population of B cells expressing germline encoded antibodies that become pathogenic merely by class switching to IgG in a pro-inflammatory milieu. We demonstrate here that IgM, DNA-reactive antibodies obtained from lupus patients that are unmutated and display polyreactivity can bind to isolated glomeruli and exhibit neurotoxic potential. Thus, the IgM polyreactive repertoire in SLE includes antibodies that may acquire pathogenic function merely by undergoing class-switch recombination to become IgG antibodies.

Tubulin is a neuronal target of autoantibodies in Sydenham's chorea.

Sydenham's chorea is a CNS disorder and sequela of group A streptococcal infection where deposition of Abs in brain may result in movement and neuropsychiatric abnormalities. We studied human mAbs 24.3.1, 31.1.1, and 37.2.1 derived from chorea and selected for cross-reactivity with group A streptococci and brain Ags. Our novel findings reveal that Sydenham's chorea mAbs target a 55-kDa brain protein with an N-terminal amino acid sequence of MREIVHLQ corresponding to beta-tubulin. Chorea mAb specificity for purified brain tubulin was confirmed in ELISA and Western immunoblot, and significant levels of anti-tubulin IgG were found in acute chorea sera and cerebrospinal fluid. Lysoganglioside G(M1) inhibited binding of chorea mAbs to tubulin and mAb reactivity with human caudate and putamen brain sections was blocked by anti-tubulin mAb. The chorea mAbs labeled both intra- and extracellular Ags of a neuronal cell line providing evidence suggesting mimicry between intracellular brain protein tubulin and extracellular lysoganglioside. In addition, chorea mAb 24.3.1 and acute chorea sera induced calcium/calmodulin-dependent protein kinase II activity in human neuronal cells. Nucleotide sequence analysis of the chorea mAb V(H) genes revealed that mAb 24.3.1 V(H) gene was encoded by the V(H)1 germline gene family which encodes other anti-ganglioside V(H) genes associated with motor neuropathies. mAb recognition of tubulin and the neuronal cell surface with initiation of cell signaling and dopamine release supports an emerging theme in autoimmunity whereby cross-reactive or polyreactive autoantibodies against intracellular Ags recognize cell surface epitopes potentially leading to disease.

Identification of HIV gp41 membrane proximal epitope tetramer reactive populations of B220+ B cells and B220lo/neg basophils in gp41 peptide-immunized mice (45.3)

Abstract 2F5 is a rare, neutralizing human mAb specific for the HIV gp41 membrane proximal region (MPER). Recently, we showed that 2F5 is a cardiolipin-binding, polyreactive autoantibody. To determine if MPER antibodies (Ab) are controlled by tolerance mechanisms, BALB/c mice were immunized with HIV-1 gp140 envelope or gp41 peptides. Immunized mice were screened for serum MPER Ab and for 2F5 antigen-specific B cells using 2F5 epitope B cell tetramers. Immunization in Ribi adjuvant did not induce anti-MPER Ab, whereas similar immunizations in TLR9, an adjuvant designed to break tolerance, did. Interestingly, in mice immunized with gp41 peptide, but not gp140, we found two distinct populations of 2F5 tetramer+ cells in bone marrow, spleen, and blood: a polyclonally-induced B220hi B-cell population that bound 2F5 tetramers via their surface Ig and a B220lo/neg cell population induced by immunization. Histological and cell surface phenotypic analysis of the B220lo/neg 2F5 tetramer+ cells indicated these were primarily basophils that bound 2F5 tetramers via MPER Ab bound to FcR. At present, the immunoregulatory significance of these distinct anti-MPER reactive populations is unknown, but their further characterization and the types of Ab they produce/bind should help in delineating why induction of neutralizing anti-MPER Ab is not achieved, despite the generation of a robust non-neutralizing MPER B cell and Ab response.

Spreading of antibody reactivity to non-thyroid antigens during experimental immunization with human thyroglobulin

Intermolecular spreading of antibody reactivity has been implicated in the evolution of autoimmune disease. In this study, spreading of antibody reactivity to non-thyroid autoantigens after experimental immunization with thyroglobulin (Tg) was investigated. For this purpose, two rabbits were injected with human Tg six times (stages 1-6) every 3 weeks. Animals were also bled before priming. Antisera were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity to several non-thyroid antigens: bovine serum albumin (BSA), native DNA (nDNA), human myosin, human globular (G) and filamentous (F) actin and porcine tubulin. Tg-immunized animals developed the following serological reactivity pattern: (a) high reactivity to myosin from stage 2 onward, (b) significant reactivity to F-actin, remaining high up to stage 6, (c) reactivity to BSA with a peak at stage 3, (d) a small increase of reactivity to G-actin at stage 3 and (e) no increase of reactivity to nDNA and tubulin. The study of affinity-purified anti-Tg antibodies and the use of competitive assays revealed that reactivity to F-actin was not due to cross-reaction with Tg. On the contrary, reactivity to myosin during the first stages of immunization was due to cross-reaction with Tg, while at stage 6 it became myosin-specific. Reactivity to BSA at stage 3 was also due to cross-reaction with Tg. We conclude that at least part of the induced anti-Tg antibodies may result from the expansion of B cell clones producing polyreactive natural autoantibodies, and polyreactivity of anti-Tg antibodies during the first stages of Tg-immunization may be responsible for the intermolecular spreading of antibody response.