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Autoimmune Disease Unit (S.M.M., P.N.P., Y.M., C.-R.C., A.M., H.A.A., B.R.), Cedars-Sinai Research Institute and University of California, Los Angeles, School of Medicine, Los Angeles, California 90048; and Department of Medical Gene Technology (Y.N.), Molecular Medicine Unit, Atomic Bomb Disease Institute, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8523, Japan
Address all correspondence and requests for reprints to: Sandra M. McLachlan, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048. E-mail: mclachlans{at}cshs.org.
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Introduction |
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The relationship between Graves’ disease and Hashimoto’s thyroiditis has been debated for decades. Although initially considered to be two separate diseases, the present view is that they represent the opposite sides of the same coin, or the two ends of a spectrum. On the other hand, whole-genome scanning studies in humans have revealed distinct differences between loci linked to, or associated with, these two autoimmune thyroid diseases (for example, Ref. 1). Moreover, animal models of Graves’ disease and Hashimoto’s thyroiditis are studied as two distinct entities. Not surprisingly, the pathophysiological relationship between TSHR, TPO, and Tg autoantibodies remains an enigma. For example, why do TPO and Tg autoantibodies arise in Graves’ disease? Do TSHR, TPO, and Tg autoantibodies arise independently or through intermolecular spreading, and if the latter, what is the primary antigen? Is the TSHR the autoantigen associated with lymphocytic infiltration in Graves’ disease?
Thyroid-stimulating antibodies, the proximal cause of Graves’ hyperthyroidism, arise from the breakdown in self-tolerance to the TSHR, a G protein-coupled receptor with seven transmembrane-spanning domains (reviewed in Ref. 2). However, the autoantigen that drives the immune response in Graves’ disease is not the full-length receptor but the A-subunit (3, 4), an ectodomain component that is shed after intramolecular cleavage of the receptor (reviewed in Ref. 2). Recently, we generated transgenic mice with the human A-subunit targeted to the thyroid gland (5). The founder transgenics were crossed with BALB/c mice, a strain that is susceptible to immunization with adenovirus expressing either the TSH holoreceptor or its A-subunit (4, 6). Unlike wild-type littermates, the transgenics failed to develop T cell responses or TSHR antibodies after low-dose A-subunit adenovirus (A-subunit-Ad) immunization. However, tolerance to the human A-subunit was partially overcome by immunization with high doses of adenovirus expressing the A-subunit or the holoreceptor (5).
Development of tolerance is a complex process that includes central and peripheral mechanisms acting in concert to eliminate self-reactive lymphocytes (7). T cell deletion by central tolerance may not eliminate all self-reactive cells. Another potent mechanism involves regulatory T cells (Treg), such as naturally occurring CD25+ CD4+ T cells or CD8+ CD122+ cells, that control autoreactive effector T cells in the periphery (8, 9). In the present study, we used A-subunit transgenic animals to probe the influence of A-subunit transgene expression levels and Treg depletion on the immune response to TSHR-Ad immunization. We report that Treg are a major factor in the intermolecular spreading of the immune response from the TSHR to TPO and Tg as well as in the shift from hyperthyroidism to full-blown Hashimoto’s thyroiditis with massive thyroid lymphocytic infiltration and hypothyroidism. These findings provide novel insight into the enigmatic balance between hyperfunction and thyroid destruction in human Graves’ disease.
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Materials and Methods |
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Human A-subunit protein expression in transgenic thyroids
Intrathyroidal expression of human TSHR A-subunit was previously demonstrated by RT-PCR (5). To examine A-subunit protein expression, we used a murine anti-pentahistidine (anti-5H) antibody (QIAGEN, Valencia, CA) to detect the C-terminal 6-histidine (6H) tag encoded by the transgene. Immunohistochemistry was performed by the Research Animal Diagnostic Laboratory (University of Missouri, Columbia, MO) as follows: Paraffin-embedded sections of murine thyroid tissue were dewaxed and rehydrated followed by heat-induced epitope retrieval (steam at 97 C for 30 min in 10 mM citrate buffer, pH 6.0). Sections were cooled and treated with 3% H2O2 (to quench endogenous peroxidase) followed by anti-5H (1:100), biotinylated rabbit antimouse IgG, and horseradish peroxidase-streptavidin (Dako North America, Inc., Carpinteria, CA). Color was developed with 3,3' diaminobenzidine (Dako), and H2O2 and the sections were counterstained with Mayer’s hematoxylin.
The concentrations of human A-subunit protein were measured in thyroid extracts prepared by homogenization (three glands per transgenic line or wild-type littermates) in buffer containing protease inhibitors (Roche Applied Science, Indianapolis, IN) and the 14,000 x g supernatant retained. We modified our ELISA for detecting TSHR antibodies in mouse sera (10) to estimate human A-subunit concentrations in these extracts. To distinguish the mouse and human A-subunits, anti-5H was used to detect the 6H-tagged transgenic protein. Duplicate supernatant aliquots (1:5 to 1:30) were preincubated with an equal volume of biotinylated anti-5H (QIAGEN; 1:1000). In the absence of thyroid extract, this anti-5H dilution yielded an OD of about 1.00 on ELISA wells coated with A-subunit (1 mg/ml) expressed in eukaryotic cells and purified by affinity chromatography (11). Preincubated test samples were transferred to A-subunit-coated wells and, after further incubation and washing, subsequently exposed to horseradish peroxidase-streptavidin (BD Biosciences, San Jose, CA). Color was developed with o-phenylene diamine and H2O2 and stopped with H2SO4, and OD values were read at 490 nm. A-subunit concentrations were expressed as milligrams per thyroid gland estimated from a standard curve generated in the same assay using non-plate-bound A-subunit (0–20 mg/ml).
Tg concentrations were measured by a similar inhibition assay using ELISA wells coated with murine (m)Tg (1 mg/ml) and a polyclonal mouse antihuman Tg that cross-reacts with mTg (generously provided by Dr. Terry F. Davies, Mount-Sinai Medical Center, New York, NY; and Dr. Yaron Tomer, University of Cincinnati, Cincinnati, OH). The standard curve, generated using 0–20 mg/ml mTg and the polyclonal anti-Tg at 1:1500, was used to calculate mTg concentrations. Total protein concentrations were determined by Bradford assay (12).
Adenovirus immunization
RGD-Ad encoding either the TSH holoreceptor or the A-subunit (5) were used in this study and are referred to as TSHR-Ad. The same adenovirus stock was always used in an individual experiment. Control immunizations were performed with adenovirus lacking an insert [control adenovirus (Con-Ad)] (13). Viruses were propagated in HEK293 cells and purified by CsCl density gradient centrifugation, and viral particle concentration was determined by absorbance at 260 nm (14). A-subunit transgenic mice and wild-type littermates (7–10 wk old) were immunized three times at three-weekly intervals with high doses of TSHR-Ad or Con-Ad (
1010 particles per injection). Blood was drawn 1 wk after two injections, and animals were euthanized 1 month after the third injection to harvest blood and thyroid glands. Animal studies were approved by the Institutional Animal Care and Use Committee at Cedars-Sinai Medical Center and performed with the highest standards of care in a pathogen-free facility.
Depletion of Treg
Rat hybridomas PC61 (anti-CD25) and TMß1(15) (anti-CD122; generously provided by Dr. K. Yui, Nagasaki University, Nagasaki, Japan; and Dr. T. Tanaka, Osaka University, Osaka, Japan, respectively) were injected into nude mice to induce ascites. The antibodies were purified on HiTrap protein G HP columns (Amersham, Piscataway, NJ) and their efficacy tested in BALB/c mice (Charles River Japan Laboratory Inc., Tokyo, Japan). Splenocytes from untreated and injected mice were compared by FACScan flow cytometry (CellQuest software; BD Biosciences, Mountain View, CA) using the following antibodies: fluorescein isothiocyanate (FITC)-anti-CD4 (H129.19) and phycoerythrin-anti-CD25 (7D4) (BD Biosciences, San Jose, CA) or FITC-conjugated-anti-CD122 (5H4; eBioscience, San Diego, CA) and phycoerythrin-conjugated anti-CD8 (53–6.7, BD Biosciences). Four days after ip injection of anti-CD25 (500 µg PC61), CD25+ CD24+ T cells were reduced from 8 to 2.1% (supplemental Fig. S1, a and b, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org) in accordance with previous observations (16). After injecting anti-CD122 (TMß1, 250 µg), CD122+, CD8+ T cells were reduced from 17.5 to 2.9% (supplemental Fig. S1, c and d). From these data, Treg depletion was performed by ip injections of 500 µg/mouse anti-CD25 or 250 µg/mouse anti-CD122 4 d before adenovirus immunization. These studies were conducted according to the principles and procedures in the Guideline for the Care and Use of Laboratory Animals, Nagasaki University.
Serum T4, thyroid histology, and TSH
Total T4 was measured in undiluted mouse serum (25 ml) by RIA using a kit (Diagnostic Products Corp., Los Angeles, CA). Thyroids were fixed in buffered formaldehyde (pH 7.4) and paraffin-embedded, and serial sections were stained with hematoxylin and eosin (Research Animal Diagnostic Laboratory, University of Missouri, Columbia, MO). Serial thyroid sections were examined without knowing the immunization employed or the origin (transgenic or wild type) of the tissue. Lymphocytic infiltration was assessed as a percentage of the tissue involved. TSH levels in some mice were determined (with a fee for service) by Dr. Roy Weiss (Thyroid Unit, University of Chicago, Chicago, IL) in undiluted serum (50 µl) by RIA (17).
TSHR antibodies
TSHR antibody levels were measured by inhibition of TSH binding to the TSHR (abbreviated TBI) using a commercial kit according to the manufacturer’s protocol (Kronus, Boise, ID). In brief, aliquots (25 µl) of mouse serum were incubated with detergent-solubilized TSHR; 125I-TSH was added and the TSHR-antibody complexes were precipitated with polyethylene glycol. TBI values were calculated from the formula: [1 – (TSH binding in test serum – nonspecific binding)/(TSH binding in control serum – nonspecific binding)] x 100.
Autoantibodies to mTg
Autoantibody binding to mTg was measured using ELISA wells coated with mTg (1 µg/ml); the positive control was a cross-reacting polyclonal mouse antihuman Tg (mTg and antibody from Drs. Davies and Tomer; see above). Test sera were diluted 1:100, antibody binding was detected with horseradish peroxidase-conjugated mouse anti-IgG (Sigma-Aldrich, St. Louis, MO), and the signal was developed with o-phenylenediamine and H2O2. The data are expressed as OD 490 nm.
Autoantibodies to mTPO
The cDNA for mTPO (18) (provided by Dr. S. Ohtaki, Japan) was transferred to the vector pHMCMV6 (19). Expression of the mTPO-pHMCV6 plasmid was tested by transiently transfecting COS-7 cells using Fugene HD (Roche) and antihuman TPO induced in mice using adenovirus (20) that cross-reacts with mouse TPO. Antibody binding was detected using FITC-conjugated affinity-purified goat antimouse IgG (Caltag Laboratories, Burlingame, CA) and analysis by flow cytometry. Cells stained with propidium iodide (1 µg/ml) were excluded from analysis. Sera (diluted 1:50) from immunized transgenics and wild-type littermates were tested for mTPO binding in the same way. Data are expressed as percent positive cells in the gated fraction (M2).
Statistical analyses
The statistical significance of differences between the magnitude of responses in multiple groups was determined by ANOVA and testing between two groups by Mann Whitney rank sum test or, when normally distributed, by Student’s t test.
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Results |
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, top panels). Although the TSHR holoreceptor is expressed on the basal surface of the thyrocyte, the soluble A-subunit is a secreted protein (21). Thyroids from 51.9 transgenics have a much lower A-subunit expression, similar to that in wild-type mice (Fig. 1A, bottom panels). In thyroid extracts, the highest A-subunit protein level (measured by ELISA inhibition) was in transgenic line 50.6, followed by line 60.6, with minimal or undetectable levels in 51.9 and wild-type mice (Fig. 1B). In contrast, as controls, thyroid concentrations of mTg and total protein were similar in all three transgenic lines and wild-type littermates. The differences in human A-subunit expression among transgenic lines were unrelated to transgene copy number, with two copies present in 50.6 transgenics and five copies in the 51.9 and 60.6 transgenics. In subsequent studies, we focused on human TSHR A-subunit transgenic lines 50.6 and 51.9, termed high (Hi) and low (Lo) expressors, respectively.
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). As expected, wild-type littermates had a robust TBI response 1 wk after the second injection and 1 month after the third injection (Fig. 2B). In contrast, Hi-expressor transgenics remained tolerant at both time intervals, even after Treg depletion with anti-CD25 (Fig. 2C). Unlike the Hi-expressor transgenics, TSHR-Ad immunization of Lo-expressors did induce TBI activity; Treg depletion using anti-CD25 or anti-CD122 did not alter the TSHR antibody response (Fig. 2D). Although some responses tended to be higher in some CD25-treated compared with untreated transgenics, the differences were not significantly different.
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Thyroid function and pathology in Lo and Hi A-subunit expressor transgenics
TSHR antibodies detected in the TBI assay may also have thyroid stimulatory activity with resultant thyrotoxicosis. We, therefore, assessed thyroid function by determining serum T4 levels in the mice immunized with TSHR-Ad. As expected in wild-type littermates, high-dose TSHR-Ad induced elevated serum T4 in about one third of mice (Fig. 3A). Consistent with the absence of TSHR antibodies (Fig. 2
), all Hi-expressor transgenics remained euthyroid, even after Treg depletion with anti-CD25 (Fig. 3B). Despite detectable TBI activity, no Lo-expressor transgenics developed hyperthyroidism. Surprisingly, 1 wk after the second immunization, serum T4 levels were subnormal in all Lo-expressors pretreated with anti-CD25 (Fig. 3C). Some recovery in T4 levels was evident at euthanasia, 4 wk after the third immunization. However, four of seven mice pretreated with anti-CD25 had extremely low or undetectable serum T4 levels (Fig. 3C). Because of these unusual findings, we measured serum TSH levels in the Lo-expressors and wild-type littermates for which sufficient serum was available at euthanasia. TSH was markedly elevated in both mice with very low T4 levels (Fig. 3D, arrows), confirming their hypothyroid status.
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, A and B). Hyperthyroid wild-type mice developed thyroid hyperplasia without infiltrating lymphocytes (Fig. 4C), as observed by ourselves and others (4, 6, 22, 23). Some Lo-expressor transgenics pretreated with anti-CD122 before TSHR-Ad immunization had modest lymphocytic infiltrates (Fig. 4D). Most striking, however, thyroids of hypothyroid Lo-expressor transgenics Treg depleted with anti-CD25 had massive lymphocyte infiltrates, encompassing much of the thyroid (Fig. 4, E and F). These findings are consistent with a shift from Graves’ disease to Hashimoto’s thyroiditis.
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, right), all of whom were hypothyroid at the time of euthanasia (Fig. 3C). Smaller infiltrates (less than 20% of the gland) were present in euthyroid animals, including some transgenics pretreated with anti-CD122, as well as in one wild-type mouse depleted of Treg using anti-CD25.
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). Similarly, autoantibodies to mTPO were positive in four of six anti-CD25-treated transgenics (Fig. 6B).
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Discussion |
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Immunization of the transgenic lines with TSHR-Ad indicated that Hi-expressor mice had a suppressed or absent immune response compared with Lo-expressors. These data are consistent with the Hi-expressors having a greater degree of central tolerance, a process in which self-reactive T cells, which bind with high affinity to peptides from self-antigens expressed in the thymus, are deleted. Intrathymic expression of the TSHR has been reported for humans (24, 25) and rats (26), but A-subunit mRNA was undetectable in thymic tissue from Hi-expressor mice (not shown). Although some self-antigens, for example Tg (27), can be studied in thymic tissue (predominantly nonexpressing thymocytes), others are studied in thymic medullary epithelial cells (28). We cannot, therefore, exclude a role for peripheral tolerance in the A-subunit transgenics. However, as in mice transgenic for hen egg lysozyme (29), it is likely that high peripheral expression of the human A-subunit correlates with increased central tolerance (and low peripheral expression with decreased tolerance) to the TSHR. It has recently been recognized that thymic expression of several tissue-restricted antigens varies considerably between individuals (28). Therefore, in humans, variability in intrathymic expression of TSHR could play a role in the breakdown of tolerance in Graves’ disease.
Because Treg, such as naturally occurring CD25+ CD4+ T cells and CD122+ CD8+ T cells could also contribute to tolerance to TSHR immunization in the A-subunit transgenic animals, we probed the influence of Treg depletion on the immune response to TSHR-Ad immunization. Immunization with a high dose of TSHR-Ad alone or preceded by anti-CD25 Treg depletion failed to induce TSHR antibodies in Hi-expressor transgenics. In contrast, Lo-expressors developed TSHR antibodies regardless of Treg depletion. Therefore, breaking tolerance is inversely related to the extent of A-subunit transgene expressed in the thyroid, and Treg do not appear to play an important role in this process.
The above studies on tolerance led to serendipitous findings providing novel insight into the role of Treg in the progression of Graves’ hyperthyroidism to severe autoimmune thyroiditis and hypothyroidism. A clinical relationship between these two diseases has long been recognized. For example, without surgical or radioiodine thyroid ablation, the long-term natural course of hyperthyroid Graves’ disease is not uncommonly hypothyroidism (30). As in Hashimoto’s thyroiditis, diffuse lymphocytic infiltration is present in Graves’ thyroids, although typically much less extensive and without thyroid follicle destruction. Although considerable progress has been made in understanding the pathogenesis of Graves’ disease and Hashimoto’s thyroiditis, the mechanism underlying a shift in the balance between these two phenotypes remains enigmatic.
In experimental animals, Graves’ disease and Hashimoto’s thyroiditis are generally studied as separate diseases, the former induced by immunization with the TSHR, the latter with Tg or, less commonly, TPO. Unlike in human disease, Graves’ hyperthyroidism induced in mice has previously not been associated with significant thyroid lymphocytic infiltration (4, 6, 22, 23) or the appearance of TPO and Tg autoantibodies. With minimal or no lymphocytic infiltration, it is self-evident that there are no reports of progression of hyperthyroidism to hypothyroidism in these mice. Lymphocytic thyroiditis is readily induced using adjuvant combined with self protein, murine Tg (31) or mTPO (32). Despite extensive lymphocytic infiltration, in most studies, the animals remain euthyroid. Indeed, transgenic mice with thyroid-restricted expression of the chemokine CCL21 develop massive lymphocytic (B and T cell) thyroid infiltration without thyroid autoantibodies, and thyroid function remains unaffected (33). Targeting TPO by immunizing with a particular mTPO peptide (34) or by expressing a pathogenic TPO-specific T cell receptor in transgenic mice lacking normal T cells or B cells (Rag knockout) (35) can cause thyroiditis and hypothyroidism. However, until the present report, no animal model has included all the pathological features of human autoimmune thyroid disease, namely hyperthyroidism, massive lymphocytic infiltration leading to hypothyroidism, and autoantibody spreading from the TSHR to TPO and Tg, the other major thyroid-specific autoantigens.
The present study suggests that although Treg do not appear to be responsible for tolerance in our A-subunit transgenic mice, they (particularly CD25+ Treg) are the pathogenetic key to the shift in the balance from Graves’ hyperthyroidism to Hashimoto’s hypothyroidism. Previous studies have demonstrated a role for CD4+CD25+ Treg depletion in experimentally induced thyroiditis. Tg immunization of BALB/c mice, normally resistant to induction of thyroiditis, develop thyroiditis in conjunction with CD25 Treg depletion (36, 37). Mild thyroiditis was induced by CD25+ T cell depletion before immunization with TSHR-expressing adenovirus in thyroiditis-susceptible C57BL/6 (but not BALB/c) mice (16). Part of this strain difference may involve the genetically controlled thymic development of Treg (CD4+CD25+FoxP3+), with higher percentages in BALB/c than C57BL/6 mice (38). In thyroiditis-susceptible NOD mice expressing a human HLA-DR3 transgene, anti-CD25 treatment enhances iodide-induced thyroiditis (39). However, it should be emphasized that none of these studies involving Treg depletion led to severe thyroiditis or to hypothyroidism. In contrast, after CD25 Treg depletion, all Lo-expressor mice studied developed hypothyroidism after the second immunization (Fig. 3C). Moreover, all four mice whose hypothyroidism persisted 1 month after the final immunization had extensive thyroiditis encompassing 60–80% of the gland.
Besides inducing extensive thyroid lymphocytic infiltration and hypothyroidism in Lo-expressor A-subunit transgenics, immunization had another unexpected result that mimics human thyroid autoimmune disease, namely spreading of the humoral autoantibody response from the TSHR to other thyroid-specific autoantigens. We provide a hypothesis to explain these intriguing findings (Fig. 7). Unimmunized wild-type and transgenic mice are tolerant to the TSHR, TPO, and Tg (all self murine proteins) and transgenics are tolerant to the human A-subunit. After immunization with human TSHR-Ad, wild-type and transgenic mice develop T cells that recognize human TSHR peptides. Presumably, cross-reactivity to mouse TSHR peptides is limited or absent. The immune response is also restrained by Treg. Therefore, lymphocytes rarely infiltrate the thyroid in mice of this genetic background (BALB/c). However, after Treg depletion (particularly CD25+ T cells), restraint on the immune response is diminished. In transgenics (but not wild-type mice), lymphocytes home to the thyroid, the source of their target peptides, the human A-subunit. The infiltrating lymphocytes (likely including cytotoxic cells and generating cytokines) cause thyrocyte damage that can lead to overt hypothyroidism. Release of thyroid antigens in this inflammatory milieu breaks tolerance to other thyroid antigens. Thus, in the Lo-expressor A-subunit transgenics, CD25 (but not CD122) Treg depletion was accompanied by strong autoantibody responses to mTg and mTPO. Previously, very low-level mTg autoantibodies were observed in a single human TSHR-DNA-vaccinated DR3– NOD mouse (40), a background susceptible to developing spontaneous thyroiditis and Tg autoantibodies. Consequently, our findings provide the first unequivocal evidence for intermolecular autoantibody spreading in thyroid autoimmunity.
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What evidence is available of a role for Treg in human thyroid autoimmunity? In humans, the number and function of Treg are still unclear, depending on the Treg markers and assays employed as well as the disease. Abundant Treg were found infiltrating the thyroid gland of Graves’ patients in one study, but the suppressor function of peripheral Treg was decreased (42). In another study, intrathyroidal Treg were reduced compared with those in peripheral blood, possibly because of increased apoptosis (43). Despite the limited number of studies (and in some cases the limited number of patients investigated), these data are consistent with our findings for the association between thyroiditis and Treg in mice and (incidentally) with the early studies of Volpe and Iitaka (44) concerning a suppressor T cell defect. Future studies on the evolution of Graves’ disease into Hashimoto’s thyroiditis with hypothyroidism will be of interest to determine whether this shift is accompanied by an alteration in Treg number or function.
In summary, the present study provides the first description of a complete animal model of autoimmune thyroid disease mimicking all the clinical and pathological features of human disease. Moreover, it describes an immunological mechanism whereby induction of thyroid-stimulating antibodies and Graves’ hyperthyroidism can be diverted to spreading of the immune response to endogenous thyroid autoantigens (mTPO and mTg) with extensive lymphocytic infiltration and hypothyroidism. Our data, taken together with other studies on experimental thyroiditis, support the clinical observation that Hashimoto’s thyroiditis in humans is only rarely associated with TSHR autoantibodies, the antithesis of Graves’ disease in which autoantibodies to Tg and TPO are commonly present. This finding has two important clinical implications. First, in Graves’ disease, TPO and Tg autoantibodies are secondary to the immune response to the TSHR. Second, lymphocytic infiltration in Graves’ disease is likely to reflect spreading of the immune response to TPO and to Tg.
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Acknowledgments |
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Footnotes |
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Disclosure Summary: The authors have nothing to disclose.
First Published Online September 6, 2007
Abbreviations: A-subunit-Ad, A-subunit-adenovirus; Con-Ad, control adenovirus; 5H, pentahistidine; m, murine; TBI, TSH binding to the TSH receptor; Tg, thyroglobulin; TPO, thyroid peroxidase; Treg, regulatory T cells; TSHR, TSH receptor.
Received July 26, 2007.
Accepted for publication August 24, 2007.
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knockout mice does not enhance TSH receptor antibody production after naked DNA vaccination. Endocrinology 143:1182–1189This article has been cited by other articles:
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  X. G Vos, N. Smit, E. Endert, J. G P Tijssen, and W. M Wiersinga Variation in phenotypic appearance of Graves' disease: effect of genetic anticipation and duration of complaints Eur. J. Endocrinol., July 1, 2009; 161(1): 113 - 118. [Abstract] [Full Text] [PDF]
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 A. V. Misharin, Y. Nagayama, H. A. Aliesky, B. Rapoport, and S. M. McLachlan Studies in Mice Deficient for the Autoimmune Regulator (Aire) and Transgenic for the Thyrotropin Receptor Reveal a Role for Aire in Tolerance for Thyroid Autoantigens Endocrinology, June 1, 2009; 150(6): 2948 - 2956. [Abstract] [Full Text] [PDF]
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S. H. Wang, G.-H. Chen, Y. Fan, M. Van Antwerp, and J. R. Baker Jr. Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Inhibits Experimental Autoimmune Thyroiditis by the Expansion of CD4+CD25+ Regulatory T Cells Endocrinology, April 1, 2009; 150(4): 2000 - 2007. [Abstract] [Full Text] [PDF]
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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CD25) or 
, wild-type mice (B); 
, Hi-expressor transgenics (C); speckled circles, Lo-expressor transgenics (D). The shaded area represents the mean ± 2 SD TBI levels for Con-Ad-immunized wild-type mice.
