Experimental autoimmune thyroiditis in mice chronically treated from birth with anti-IgM antibodies

The role of T lymphocytes in the pathogenesis of experimental autoimmune thyroiditis in mice is well established while the role of B lymphocytes is unclear. Mice with thyroid lesions have thyroglobulin antibodies whereas these antibodies can occur in mice immunized with Tg that do not develop thyroid lesions. To determine whether thyroglobulin antibodies are necessary for the development of the thyroid infiltrates with mononuclear cells, which are characteristic for experimental autoimmune thyroiditis, AKR mice chronically treated from birth with goat anti-mouse IgM antibodies were immunized with mouse thyroglobulin in Freund's complete adjuvant when they were 7 weeks old. Control mice, similarly immunized, were chronically injected from birth with normal goat gamma-globulin. Three weeks after immunization, all mice were sacrificed, thyroglobulin antibodies in the serum were measured by hemagglutination assay and enzyme-linked immunosorbent assay, and thyroid pathology was assessed. The serum concentration of IgG and IgM, the percentage of B and T lymphocytes in the spleen (flow cytometry), and the in vitro proliferative response of spleen lymphocytes to stimulation by PHA, LPS, and Tg were also measured. All mice treated with anti-IgM antibodies did not have detectable thyroglobulin antibodies but 63% of these mice and 88% of control mice (all of which had thyroglobulin antibodies) had thyroid lesions. Mice treated with anti-IgM antibodies that did not have thyroid lesions had a more pronounced depression of B lymphocytes than similarly treated mice that had thyroid lesions. These experiments suggest that thyroglobulin antibodies are not necessary for the development of thyroid infiltrates with mononuclear cells. B lymphocytes could still participate in the production of experimental autoimmune thyroiditis by presenting thyroglobulin to helper T lymphocytes.     

Production of anti-thyroglobulin antibody by blood lymphocytes from patients with autoimmune thyroiditis, induced by the insolubilized autoantigen

Mononuclear cells (MNC) from the blood of patients with autoimmune thyroiditis cannot be stimulated in vitro with the autoantigen thyroglobulin (Tg). However, when Tg was coupled with a solid matrix, MNC from 13 of 19 patients tested responded to this insolubilized Tg (i-Tg) by generating cells which produced IgG anti-Tg antibodies, as demonstrated in a "spot ELISA". This response was specific not only in the assay phase but also in the induction phase, because 1) cultures with insolubilized ovalbumin, bovine Tg, or porcine Tg were negative, and 2) i-Tg did not induce polyclonal IgG production. Generation of anti-Tg-producing cells was completely abrogated when free Tg was added to i-Tg cultures. Stimulation with i-Tg after separation of T and non-T MNC revealed that: 1) the response was independent of T cells; 2) in three of the six patients whose MNC did not respond, removal of T cells did result in anti-Tg production; 3) re-addition of the T cells--but not of irradiated T cells--abrogated the response in two of these three patients; and 4) preincubation of monocytes with i-Tg resulted in suboptimal anti-Tg production. These findings indicate that B cells from patients with autoimmune thyroiditis can be specifically stimulated with Tg coupled with a solid matrix, resulting in generation of anti-Tg-producing cells. The essence of this mode of presenting the antigen would appear to be the cross-linkage of surface immunoglobulin. The response was T cell-independent; in some patients, however, the presence of a radiosensitive suppressor T cell population can be demonstrated.     

Inhibition of experimental autoimmune thyroiditis in mice by anti-I-A antibodies

Experimental autoimmune thyroiditis is induced in mice by immunization with thyroglobulin emulsified in Freund's complete adjuvant. The disease is characterized both by thyroid infiltration with mononuclear cells and by circulating thyroglobulin antibodies. The magnitude of the thyroid infiltration and the titer of thyroglobulin antibodies are controlled by genes in the I-A subregion of the major histocompatibility complex (H-2). We investigated the in vivo effect of monoclonal anti-Ia antibodies on experimental autoimmune thyroiditis in susceptible mice. Antibodies were given around the time of immunization, later after immunization, and to mice with established disease. Monoclonal antibody produced by the hybridoma line 10-3.6 (anti-I-Ak, s, u, v, z, f) completely prevented both production of thyroglobulin antibodies and thyroid infiltrates, when given shortly before or at the time of antigen administration. This effect was dose-dependent and this monoclonal antibody decreased the severity of the disease when given after the antigen challenge but did not fully suppress established thyroiditis. The same antibody markedly decreased the number of B lymphocytes in the spleen and decreased the thyroglobulin-induced spleen cell proliferation when either given in vivo or added in vitro to cell cultures. Antibodies produced by the hybridoma line 11.2.12 (anti-I-Ak) did not show an inhibitory effect on the disease. These experiments suggest that in this model of murine thyroiditis anti-Ia antibodies act on antigen-presenting cells. Furthermore, only one monoclonal antibody, anti-Ia, suppressed the immune response to thyroglobulin, suggesting a possible role for the isotype and specificity of anti-Ia antibody.     

Suppression in experimental autoimmune thyroiditis: the role of unique and shared determinants on mouse thyroglobulin in self-tolerance.

Previous studies have shown that T cells from mice genetically susceptible to experimental autoimmune thyroiditis (EAT) recognize determinants shared between mouse thyroglobulin (Tg) and heterologous Tgs. Some shared determinants are thyroiditogenic; lymphocytes from mice immunized with mouse Tg (MTg) or human Tg (HTg) and reciprocally restimulated in vitro with either Tg can transfer EAT. Studies on the mechanisms of self-tolerance have shown that pretreatment with soluble MTg suppresses in vitro proliferation to MTg and EAT induction with MTg. To determine the role of share epitopes in maintaining tolerance, mice were pretreated with soluble HTg and immunized with HTg or MTg and adjuvant. Cells from HTg-pretreated. HTg-immunized mice showed suppressed in vitro proliferative response to HTg. Following MTg immunization, the cells showed suppressed in vitro response to MTg. However, in contrast to MTg pretreatment, the subsequent development of EAT in vivo was unaltered in severity following HTg pretreatment. Thus, determinants shared between HTg and MTg can induce suppression of in vitro responses to HTg and MTg, but not inhibit the onset of thyroiditis, suggesting that T cells recognizing MTg-unique epitopes expanded to mediate thyroiditis. We conclude that recognition of both unique epitopes expanded to mediate thyroiditis. We conclude that recognition of both unique and shared epitopes on MTg are essential for the overall maintenance of self-tolerance.     

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.     

Microbial adjuvant and autoimmunity. IV. The induction of thyroid lesions in syngeneic X-irradiated mice by the transfer of spleen cells from mice immunized with thyroid extract and Klebsiella O3 lipopolysaccharide

The role of humoral and cellular immune responses in the initiation and maintenance of autoimmune thyroiditis was investigated in mice immunized with syngeneic thyroid extract and Klebsiella O3 lipopolysaccharide (KO3 LPS) as an adjuvant. The transfer of spleen cells from hyperimmunized mice to 400R-irradiated syngeneic mice produced definite lesions in the thyroid glands, whereas the transfer of immune sera failed to do so. No lesions were induced in normal intact mice by the same transfer of sera and spleen cells from hyperimmunized mice. It was suggested that the induction of thyroiditis by immunization using KO3 LPS adjuvant is primarily due to cell-mediated immunity and that pretreatment of mice by X-irradiation is essential for production of the lesions. The role of X-irradiation in the induction of thyroiditis was discussed.     

cDNA immunization of mice with human thyroglobulin generates both humoral and T cell responses: a novel model of thyroid autoimmunity

Thyroglobulin (Tg) represents one of the largest known self-antigens involved in autoimmunity. Numerous studies have implicated it in triggering and perpetuating the autoimmune response in autoimmune thyroid diseases (AITD). Indeed, traditional models of autoimmune thyroid disease, experimental autoimmune thyroiditis (EAT), are generated by immunizing mice with thyroglobulin protein in conjunction with an adjuvant, or by high repeated doses of Tg alone, without adjuvant. These extant models are limited in their experimental flexibility, i.e. the ability to make modifications to the Tg used in immunizations. In this study, we have immunized mice with a plasmid cDNA encoding the full-length human Tg (hTG) protein, in order to generate a model of Hashimoto's thyroiditis which is closer to the human disease and does not require adjuvants to breakdown tolerance. Human thyroglobulin cDNA was injected and subsequently electroporated into skeletal muscle using a square wave generator. Following hTg cDNA immunizations, the mice developed both B and T cell responses to Tg, albeit with no evidence of lymphocytic infiltration of the thyroid. This novel model will afford investigators the means to test various hypotheses which were unavailable with the previous EAT models, specifically the effects of hTg sequence variations on the induction of thyroiditis.     

Autoimmune recognition of thyroglobulin and thyroid hormones in rabbits

Two rabbits (RG-1, RG-2) were immunized with rabbit thyroglobulin (RTg) purified from thyroid glands of four other normal rabbits of the same strain, and bled serially. Antisera were obtained at different times after the first immunization and kept separately and studied. Production of anti-RTg as well as anti-thyroid hormone antibodies such as anti-thyroxine (T4) and anti-triiodothyronine (T3) antibodies was observed in both rabbits. Physicochemical parameters of anti-RTg antibodies with RTg, T4, and T3 were calculated in two selected antisera (70-day and 253-day) of each of the rabbits, using a Scatchard plot. Extraction of serial sera from both rabbits disclosed the presence of larger amounts of T3 and T4 in immune sera than in preimmune serum. Examination of pathology of thyroid glands and kidneys in both rabbits was negative for the lesions of autoimmune thyroiditis and immune nephritis. These results indicate that anti-Tg as well as anti-thyroid hormone autoantibodies can be raised without thyroid pathology in rabbit by immunization with autologous Tg.     

Autoimmune encephalomyelitis (EAE) mediated or prevented by T lymphocyte lines directed against diverse antigenic determinants of myelin basic protein. Vaccination is determinant specific

Lines of T lymphocytes reactive against the basic protein of myelin (BP) were found in previous studies to mediate experimental autoimmune encephalomyelitis (EAE) in rats. Moreover, inoculation of rats with attenuated anti-BP line cells vaccinated them against subsequent attempts to induce active EAE by injection of BP in adjuvant. In the present study, we investigated the effects of T lymphocyte lines reactive to different antigenic determinants on the BP molecule, they are the major encephalitogenic peptide (EP) determinant present on guinea pig BP (G-BP), and minor, non-EP determinants present on bovine BP (B-BP). We found that both lines of T lymphocytes could mediate EAE. Resistance to active EAE acquired by spontaneous recovery from line mediated EAE or by vaccination with attenuated cells, however, was found to be specific for the particular BP determinant. Thus, EAE may be mediated by lines of T lymphocytes reactive to different determinants on the BP molecule, but the resistance to EAE acquired by exposure to line cells is determinant specific. This suggests that acquired resistance to EAE is directed by the receptor specificity of the autoimmune anti-BP T cells.     

Prevention of experimental autoimmune thyroiditis through the anti-idiotypic network.

The central goal in the therapy of autoimmune diseases is to develop potent tools able to exert specific control of the immune response to self Ag. Anti-Id may provide such specific immunodulators because the relevance of the idiotypic network in autoimmunity is well documented. We now describe the protective immunity against experimental autoimmune thyroiditis induced exclusively by a thyroglobulin (Tg)-specific cytotoxic T cell clone and show that this down-regulation occurs through the generation of anti-Id antibodies (Ab) (Ab2Beta) which recognize the paratope of a anti-Tg mAb (Ab1) specific to the pathogenic epitope of the Tg molecule. We further analyze the various steps of the Ab responses (Ab1, Ab2, and Ab3) in terms of poly-, mono-, and autospecificities for the pathogenic epitope of the Tg molecule and for the idiotope of the related Ab.     

Perturbation of the autoimmune network. II. Immunization with isologous idiotype induces auto-anti-idiotypic antibodies and suppresses the autoantibody response elicited by antigen: a serologic and cellular analysis

We report on the humoral and cellular events following autologous immunization against an idiotype (Id62) borne on a murine monoclonal autoantibody to thyroglobulin, and their impact on the autoantibody response to thyroglobulin. BALB/c mice with a state of active auto-anti-idiotypic immunity and challenged with thyroglobulin in complete Freund's adjuvant 2 wk after the last immunization with idiotype were found to have a suppressed autoantibody response. This suppression could be adoptively transferred to syngeneic x-irradiated recipients by using whole spleen cells from idiotype-primed mice. Transfer of separate T and B lymphocyte populations proved instrumental in disecting humoral from cellular events and in establishing that whereas B cells were required for transferring an intact anti-idiotype antibody response, T cells from idiotype-primed mice were necessary to transfer suppression. These findings contribute to our understanding of the interrelationship between antigen, idiotype, and anti-idiotype in the immune response to self-antigens, and the role of certain idiotypes in regulating autoimmune responses.     

TRAIL and DR5 Promote Thyroid Follicular Cell Apoptosis in Iodine Excess-Induced Experimental Autoimmune Thyroiditis in NOD Mice

Death receptor-mediated apoptosis has been implicated in target organ destruction in patients with chronic autoimmune thyroiditis. Several apoptosis signaling pathways, such as Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), have been shown to be active in thyroid cells and may be involved in destructive thyroiditis. Thyroid toxicity of iodide excess has been demonstrated in animals fed with an iodide-rich diet, but its pathogenic role remains unclear. The effects of excessive iodine on TRAIL and its death receptor expression in thyroid were investigated. Experimental autoimmune thyroiditis (EAT) was induced by excessive iodine and thyroglobulin (Tg) in non-obese diabetic mice. The expression of TRAIL and its death receptor DR5 was detected by immunofluorescence staining. Following administration of excessive iodine alone, Tg, and excessive iodine combined with Tg, TRAIL-positive cells appear not only in follicular cells but also in lymphocytes infiltrated in the thyroid, whereas DR5-positive cells appear only in follicular cells. Large numbers of CD3-positive cells and a few CD22-positive cells were detected in thyroid. A great amount of follicular cells were labeled specifically by terminal deoxynucleotide transferase-mediated deoxynucleotide triphosphate nick-end labeling assay. Taken together, our results suggest that excessive iodine could induce TRAIL and DR5 abnormal expression in thyroid. TRAIL band with DR5 to promote follicular cells apoptosis thus mediate thyroid destruction in EAT.     

Induction of experimental autoimmune thyroiditis in mice with in vitro activated splenic T cells

Spleen cells from CBA/J or SJL mice sensitized with mouse thyroglobulin (MTg) and lipopolysaccharide (LPS) could be activated in vitro with MTg to transfer experimental autoimmune thyroiditis (EAT) to normal syngeneic recipients. EAT induced by these transferred cells was similar in incidence and severity to EAT induced by active immunization of mice with MTg and adjuvant and cells from EAT-resistant Balb/c mice could not be activated to induce EAT. The specific antigen MTg was required both for initial sensitization of the mice and for activation of spleen cells in vitro. The cells that were active in transferring EAT to mice were shown to be T cells. Removal of B cells from the cultured spleen cells had no effect on the ability of the cells to induce EAT.     

Selective induction of dendritic cells using granulocyte macrophage-colony stimulating factor,but not fms-like tyrosine kinase receptor 3-ligand,activates thyroglobulin-specific CD4+/CD25+ T cells and suppresses experimental autoimmune thyroiditis

Fms-like tyrosine kinase receptor 3-ligand (Flt3-L) and GM-CSF cause expansion of different subsets of dendritic cells and skew the immune response toward predominantly Th1 and Th2 type, respectively. In the present study, we investigated their effects on experimental autoimmune thyroiditis in CBA/J mice. Relative to mouse thyroglobulin (mTg) immunized controls, mTg-immunized mice treated with Flt3-L showed more severe thyroiditis characterized by enhanced lymphocytic infiltration of the thyroid, and IFN-gamma and IL-2 production. In contrast, mice treated with GM-CSF, either before or after immunization with mTg, showed suppressed T cell response to mTg and failed to develop thyroiditis. Lymphocytes from these mice, upon activation with mTg in vitro, produced higher levels of IL-4 and IL-10. Additionally, GM-CSF-treated mice showed an increase in the frequency of CD4(+)/CD25(+) T cells, which suppressed the mTg-specific T cell response. Neutralization of IL-10, but not IL-4, or depletion of CD4(+)/CD25(+) cells resulted in increased mTg-specific in vitro T cell proliferation suggesting that IL-10 produced by the Ag-specific CD4(+)/CD25(+) regulatory T cells might be critical for disease suppression. These results indicate that skewing immune response toward Th2, through selective activation of dendritic cells using GM-CSF, may have therapeutic potential in Th1 dominant autoimmune diseases including Hashimoto's thyroiditis.     

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.     

Breaking Tolerance in Transgenic Mice Expressing the Human TSH Receptor A-Subunit: Thyroiditis, Epitope Spreading and Adjuvant as a 'Double Edged Sword'

Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a "double-edged sword". On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the TSHR) displaying strong self-tolerance.     

IL-12 prevents tolerance induction with mouse thyroglobulin by priming pathogenic T cells in experimental autoimmune thyroiditis: role of IFN-gamma and the costimulatory molecules CD40l and CD28

Deaggregated mouse thyroglobulin (dMTg) induces tolerance to experimental autoimmune thyroiditis (EAT), a Th1-cell-mediated disease. To test whether IL-12, a potent activator of Th1 cells, can overcome tolerance induction, different doses of IL-12 were given to CBA/J mice during the critical interval of 2--3 days after dMTg administration. After challenge with MTg/LPS, dMTg/IL-12-pretreated mice showed more extensive thyroiditis than immunized controls, but comparable levels of anti-MTg and T cell proliferation. Without challenge, few MTg antibodies were produced. In contrast, pretreatment with dMTg/poly A:U or dMTg/IL-1, two other T cell activators which also interfere with tolerance induction, induced antibodies before challenge, but not more severe thyroiditis. Mice pretreated with IL-12 without dMTg developed thyroiditis comparable to immunized controls, but less severe thyroiditis than dMTg/IL-12-pretreated mice. Clearly, IL-12 not only blocked tolerance induction, but also primed antigen-specific T cells during the tolerogenic period of dMTg pretreatment, resulting in stronger thyroiditis than immunization only. Neither treatment with anti-IFN-gamma nor the use of IFN-gamma knockout mice altered the capacity of IL-12 to prevent tolerance induction. However, both anti-CD28 and anti-CD40L antibodies diminished the priming effect by dMTg/IL-12. The mechanisms of IL-12 action include priming of MTg-specific T cells and the involvement of T cell costimulatory molecules.     

Transgenic expression of Fas ligand on thyroid follicular cells prevents autoimmune thyroiditis

"Immune privilege" is defined as tissue resistance to aggression by specifically activated lymphocytes, and involves the interaction between Fas expressed on infiltrating cells and Fas ligand (FasL) constitutively expressed on the target tissue. To test whether ectopic expression of FasL on thyrocytes could prevent autoimmune aggression of the thyroid by activated lymphoid cells, three lines of transgenic mice expressing low, intermediate, and high levels of functional FasL on thyroid follicular cells were generated. Experimental autoimmune thyroiditis was induced by immunization with mouse thyroglobulin. In all of the experiments, the effects were dependent on the level of FasL expression. Low and intermediate expression had no or only weak preventive effects, respectively, whereas high FasL expression strongly inhibited lymphocytic infiltration of the thyroid. Anti-mouse thyroglobulin-proliferative and cytotoxic T cell responses, as well as autoantibody production, were diminished in transgenic mice expressing high levels of FasL relative to controls. Furthermore, in these latter mice Th1 responses to mouse thyroglobulin were profoundly down-regulated, uncovering a new potential role for FasL in peripheral tolerance to organ-specific Ags. In sum, the prevention of experimental autoimmune thyroiditis by FasL on thyrocytes is dependent on the level of FasL expression.     

H2A- and H2E-derived CD4+CD25+ regulatory T cells: a potential role in reciprocal inhibition by class II genes in autoimmune thyroiditis

We recently described a novel H2E class II-transgenic model (A(-)E(+)) of experimental autoimmune thyroiditis (EAT) that permits disease induction with heterologous thyroglobulin (Tg), but unlike conventional susceptible strains, precludes self-reactivity to autologous mouse Tg. In transgenic E(+)B10 (A(+)E(+)) mice, the presence of endogenous H2A genes is protective against H2E-mediated thyroiditis, inhibiting EAT development. The suppressive effect of H2A genes on H2E-mediated thyroiditis mirrors previous reports of H2E suppression on H2A-mediated autoimmune diseases, including EAT. The mechanism of the reciprocal-suppressive effect between class II genes is unclear, although the involvement of regulatory T cells has been proposed. We have recently reported that CD4(+)CD25(+) regulatory T cells mediate peripheral tolerance induced with mouse Tg in CBA mice. To determine whether these cells play a role in our E(+)-transgenic model, we first confirmed the existence of CD4(+)CD25(+) T cells regulating thyroiditis in E(+)B10.Ab(0) (A(-)E(+)) and B10 (A(+)E(-)) mice by i.v. administration of CD25 mAb before EAT induction. The depletion of CD4(+)CD25(+) T cells enhanced thyroiditis induction in the context of either H2E or H2A. Moreover, reconstitution of CD4(+)CD25(+) T cells from naive B10 mice restored resistance to EAT. E(+)B10 (A(+)E(+)) mice were also depleted of CD4(+)CD25(+) T cells before the challenge to determine their role in thyroiditis in the presence of both H2A and H2E genes. Depletion of CD4(+)CD25(+) regulatory T cells offset the suppression of H2E-mediated thyroiditis by H2A. Thus, these regulatory T cells may be involved in the reciprocal-suppressive effect between class II genes.     

Suppression of experimental autoimmune thyroiditis in guinea pigs by pretreatment with thyroglobulin-coupled spleen cells

Pretreatment of Strain 2 and Strain 13 guinea pigs with guinea pig thyroglobulin (GPTG) coupled to syngeneic spleen cells (GPTG-SC) suppressed the development of experimental autoimmune thyroiditis (EAT) induced by immunization with GPTG in complete Freund's adjuvant (CFA). Antibody titers to GPTG were only minimally suppressed in GPTG-SC pretreated animals. GPTG-SC also suppressed the sensitization of periotneal exudate T lymphocytes which proliferate in vitro in the presence of GPTG.