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Endocrinology Vol. 147, No. 5 2417-2422
Copyright © 2006 by The Endocrine Society

Regulation of Graves’ Hyperthyroidism with Naturally Occurring CD4+CD25+ Regulatory T Cells in a Mouse Model

Ohki Saitoh and Yuji Nagayama

Department of Medical Gene Technology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan

Address all correspondence and requests for reprints to: Dr.Yuji Nagayama, Department of Medical Gene Technology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. E-mail: nagayama{at}net.nagasaki-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Graves’ hyperthyroidism can be efficiently induced in susceptible mouse strains by repeated immunization with recombinant adenovirus coding the TSH receptor (TSHR). This study was designed to evaluate the role(s) played by naturally occurring CD4+CD25+ regulatory T cells in the development of Graves’ hyperthyroidism in resistant C57BL/6 and susceptible BALB/c mice. Depletion of CD4+CD25+ T cells rendered some C57BL/6 mice susceptible to induction of hyperthyroidism. Thus, hyperthyroidism developed in 30% of the CD4+CD25+ T cell-depleted C57BL/6 mice immunized with adenovirus expressing the TSHR A-subunit (AdTSHR289) vs. 0% of those immunized with AdTSHR289 alone. This immunological manipulation also enhanced disease severity in susceptible BALB/c mice, as reflected by a significant increase in mean T4 levels by CD4+CD25+ T cell depletion. The immunoenhancing effect of CD4+CD25+ T cell depletion appears to be attributable to an increase in thyroid-stimulating antibody production and/or a decrease in thyroid-blocking antibody synthesis, but not immune deviation to either T helper 1 or 2 cells. Interestingly, unlike BALB/c mice, some hyperthyroid C57BL/6 mice showed some intrathyroidal lymphocytic infiltration with follicular destruction. These results indicate that CD4+CD25+ T cells play a role in disease susceptibility and severity in adenovirus-TSHR-induced Graves’ hyperthyroidism. Overall, the imbalance between effector and regulatory T cells appears to be crucial in the pathogenesis of Graves’ disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GRAVES’ HYPERTHYROIDISM is a common, but unique, organ-specific autoimmune disease, in that agonistic autoantibodies against the TSH receptor [TSHR; thyroid-stimulating antibodies (TSAb)] activate the thyroid gland, inducing hyperthyroidism and diffuse hyperplasia of the thyroid gland (1). Although the presence of TSAb in the serum of patients with Graves’ hyperthyroidism has long been recognized, the mechanisms that lead to its production have remained obscure. TSAb and hyperthyroidism can be experimentally induced in mice by several immunization approaches, including genetic immunization with recombinant adenovirus coding the TSHR, as we recently reported (2, 3). In this adenovirus-TSHR model, BALB/c and C57BL/6 mice are susceptible and resistant, respectively, to development of hyperthyroidism (2, 4). However, both mouse strains develop comparable levels of anti-TSHR antibodies, yet the resistant C57BL/6 mice higher levels of blocking antibodies (4). These data indicate that TSHR-reactive T lymphocytes are present in the periphery of any mouse strain regardless of its susceptibility, but development of hyperthyroidism by adenovirus-TSHR immunization is not determined solely by the presence of these TSHR-reactive T cells. We were interested in determining how the resistance could be reversed to permit the induction of Graves’ hyperthyroidism in C57BL/6 mice. To this purpose, we focused on naturally occurring CD4+CD25+ regulatory T cells. We hoped that the outcome of these studies would shed light on the pathogenesis of Graves’ disease in humans.

CD4+CD25+ T cells develop in the thymus and the periphery and represent 5–10% of the peripheral CD4+ T cell compartment (5). These cells suppress the proliferation of effector CD4+ T cells as well as the maturation of dendritic cells (5, 6). Depletion and repletion experiments implicate this T cell subpopulation in negative regulation of several autoimmune diseases as well as tumor immunity. Thus, transfer of splenocytes depleted of CD4+CD25+ T cells into lymphopenic nude mice can trigger autoimmune destruction of a variety of tissues (7). Deficiency of CD7 and CD28 results in the loss of regulatory CD4+CD25+ T cells and allows for the spontaneous development of autoimmune thyroiditis in otherwise normal, aged C57BL/6 mice (8). A combination of CD4+CD25+ T cell depletion and antigen immunization induces autoimmune diseases, such as autoimmune gastritis, diabetes, and experimental autoimmune encephalitis (EAE), in nonlymphopenic, resistant, or tolerant mice (9, 10, 11, 12). Collagen-induced arthritis can be exacerbated by the removal of CD4+CD25+ T cells in susceptible mice (13). Tumor immunity is also enhanced by removal of CD4+CD25+ T cells (14, 15). Furthermore, disruption of a gene coding the Forkhead box 3 transcription factor 3, which is indispensable for the development and maintenance of CD4+CD25+ T cells, leads to multiorgan-specific autoimmunity in both mice (scurfy mouse phenotype) and humans (immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome) (5). Conversely, the transfer of CD4+CD25+ T cells protects or cures autoimmune diseases such as EAE, colitis, and diabetes (16, 17, 18, 19). CD4+CD25+ T cells induced by granulocyte-macrophage colony-stimulating factor or tolerogenic semimature dendritic cells suppress thyroglobulin-induced thyroiditis (20, 21).

We show in this study that CD4+CD25+ T cell depletion not only increases disease incidence in resistant C57BL/6 mice, but also enhances disease severity in susceptible BALB/c mice. Thus, the imbalance between effector and regulatory T cells appears to be a crucial factor in the pathogenesis of Graves’ disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Female BALB/c and C57BL/6 mice (6 wk old) were purchased from Charles River Japan Laboratory, Inc. (Tokyo, Japan) and were kept in a specific pathogen-free facility. All experiments were conducted at Nagasaki University in accordance with the principles and procedures outlined in the Guideline for the Care and Use of Laboratory Animals.

Immunization protocols
Construction, amplification, purification of nonreplicative recombinant adenoviruses expressing the human TSHR-A subunit (AdTSHR289; provided by Drs. Sandra M. McLachlan and Basil Rapoport), and determination of the viral particle concentration were described previously (2, 3). Mice were injected im in the quadriceps with 100 µl PBS containing 1010 particles of AdTSHR289 (d 0) on three occasions at 3-wk intervals. Groups of mice were also treated by ip injection of 500 µg/mouse anti-CD25 monoclonal antibody on d –4 in each immunization. Anti-CD25 antibody was purified from ascites of mice injected ip with hybridoma PC61 (gift from Dr. K. Yui, Nagasaki University, Nagasaki, Japan) with a HiTrap protein G HP column (Amersham Biosciences, Piscataway, NJ). Blood, spleens, and thyroid tissues were obtained 2 wk after the final immunization. Thyroid histology was examined on formalin-fixed tissue sections stained with hematoxylin and eosin (H & E).

Flow cytometry
Splenocytes were stained with fluorescein isothiocyanate-conjugated anti-CD4 (H129.19) and PE-CD25 (7D4; BD Pharmingen, San Diego, CA) and were analyzed on a FACScan flow cytometry using CellQuest software (BD Biosciences, Mountain View, CA). Note that the binding site of 7D4 on CD25 is different from that of PC61.

T4, TSAb, thyroid-blocking antibody (TBAb), and TSH binding-inhibiting antibody (TBIAb) measurements
Total serum T4 was measured with a RIA kit (SPAC T4 RIA kit, TFB, Tokyo, Japan) in duplicate. The normal range was defined as the mean ± 3 SD for control untreated mice. TSAb and TBAb activities in mouse sera were measured with FRTL5 cells as previously described (22). Briefly, for TSAb assay, the cells (5 x 104 cells/well in a 96-well culture plate) were incubated in 50 µl hypotonic Hanks’ Balanced Salt Solution containing 0.5 mM isobutylmethylxanthine, 20 mM HEPES, 0.25% BSA, and 5 µl serum for 2 h at 37 C. cAMP released into the medium was measured with a cAMP RIA kit (Yamasa, Choshi, Japan). TBAb activities were measured in the same buffer supplemented with 100 µU/ml bovine TSH (Sigma-Aldrich Corp., St. Louis, MO), and were expressed as the percent inhibition of TSH-induced cAMP generation by test sera. TBIAb values were determined using a TRAb kit (BRAHMS Diagnostica GmbH, Berlin, Germany) and 1 µl (C57BL/6) or 10 µl (BALB/c) sera.

ELISA for anti-TSHR antibodies
ELISA wells were coated overnight with 100 µl TSHR289 protein (1 µg/ml; gift from Drs. Sandra M. McLachlan and Basil Rapoport) (22) and incubated with mouse sera (1:100 dilution). After incubation with horseradish peroxidase-conjugated, subclass-specific, antimouse IgG1, IgG2a, and IgG2b (BD Pharmingen; Caltag Laboratories, Inc., Burlingame, CA), color was developed using orthophenylene diamine and H2O2 as substrate, and OD was read at 492 nm.

Cytokine assays
Splenocytes were cultured (triplicate aliquots) at 5 x 105 cells/well in a 96-well, round-bottomed plate in the presence or absence of 5 µg/ml TSHR289 protein. Four days later, the concentrations of IFN{gamma} and IL-4 in the culture medium were determined with ELISA kits (BioSource International, Camarillo, CA). Cytokine production was expressed as picograms per milliliter using standard curves of recombinant mouse cytokines.

Statistical analysis
Levels of antibodies and cytokines and incidences of hyperthyroidism were analyzed by Student’s t test or {chi}2 test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD4+CD25+ T cell-depletion induced Graves’ hyperthyroidism in some C57BL/6 mice immunized with AdTSHR289
In an attempt to reverse their resistance to induction of hyperthyroidism, C57BL/6 mice were treated with anti-CD25 monoclonal antibody. Intraperitoneal injection of 500 µg anti-CD25 antibody significantly reduced the proportion of CD4+CD25+ T cells to CD4+ T cells in spleen (6.0% to 1.7%; Fig. 1Go).


Figure 1
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  		FIG. 1. Flow cytometric analysis of CD4 and CD25 expression on splenocytes. Mice were untreated (A) or treated with 0.5 mg anti-CD25 antibody (B). Four days later, splenocytes were analyzed for CD4 and CD25 expression by FACScan, as described in Materials and Methods.

 
As previously reported (4), none of the C57BL/6 mice immunized with AdTSHR289 developed Graves’ hyperthyroidism. However, depletion of CD4+CD25+ T cells rendered some mice of this strain susceptible to hyperthyroidism induced by AdTSHR289. Thus, three of 10 (30%) CD4+CD25+ T cell-depleted mice developed elevated T4 levels (Fig. 2AGo). CD4+CD25+ T cell depletion alone did not induce an immune response to the TSHR (data not shown).


Figure 2
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  		FIG. 2. Serum T4 concentrations in mice treated with AdTSHR289 alone or in combination with anti-CD25 antibody. T4 levels were determined 2 wk after the final immunization. Data are the means of duplicate determinations. {circ}, Hyperthyroid mice; bullet, euthyroid mice. Horizontal lines indicate the normal upper limits of T4 values. *, Significant difference (P < 0.05).

 
CD4+CD25+ T cell depletion enhanced the severity of hyperthyroidism in BALB/c mice immunized with AdTSHR289
Given the aforementioned data, the effect of CD4+CD25+ T cell depletion was also studied in susceptible BALB/c mice. Similar to findings in our recent report (3), im AdTSHR289 induced disease in approximately 70% of animals in this strain (Fig. 2BGo). Although depletion of CD25+ T cells did not increase the disease induction rate, mean T4 levels in CD4+CD25+ T cell-depleted BALB/c mice were approximately twice the values in nondepleted mice (mean ± SD, 22.2 ± 13.2 vs. 11.9 ± 5.2 µg/dl; P < 0.05), indicating a significant increase in the severity of hyperthyroidism.

CD4+CD25+ T cell depletion enhanced TSAb production and/or suppressed TBAb titers, but did not affect T helper 1 cell (Th1)/Th2 balance in C57BL/6 and BALB/c mice
Anti-TSHR antibody titers and TSHR-specific cytokine secretions from splenocytes in response to in vitro stimulation with TSHR antigen were compared in CD4+CD25+ T cell-depleted and nondepleted C57BL/6 and BALB/c mice. Anti-TSHR antibodies were determined using four different methods. TSAb and TBAb assays detect stimulating and blocking antibodies responsible for hyperthyroidism and hypothyroidism, respectively. Extremely high TSAb titers were detected in all hyperthyroid C57BL/6 and some hyperthyroid BLAB/c mice, and significantly lower TBAb titers were observed in CD4+CD25+ T cell-depleted BALB/c mice compared with nondepleted mice (Fig. 3Go, A–D). TBIAb assay measures the ability of anti-TSHR antibodies to displace [125I]TSH binding to the TSHR and cannot discriminate between stimulating and blocking antibodies. TBIAb titers appeared to be higher in depleted mice than nondepleted mice, but the difference only achieved statistical significance in BALB/c mice (Fig. 3Go, E and F). Finally, ELISA was used to measure the titers of anti-TSHR antibody IgG subclasses. No significant difference was observed in antibody subclass titers (Fig. 4Go, A and B) and the ratios of IgG2 to IgG1 (Th1 to Th2; data not shown) between depleted and nondepleted mice.


Figure 3
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  		FIG. 3. TSAb, TBAB, and TBIAb values in mice treated with AdTSHR289 alone or in combination with anti-CD25 antibody. These values were determined 2 wk after the final immunization. Data are the means of duplicate determinations. {circ}, Hyperthyroid mice; bullet, euthyroid mice. *, P < 0.05; **, P < 0.01.

 

Figure 4
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  		FIG. 4. Th1/Th2 balance in mice treated with AdTSHR289 alone or in combination with anti-CD25 antibody, as assessed by antibody IgG subclass ELISA and in vitro IFN{gamma} recall assay. A and B, IgG subclass ELISA was performed with sera obtained 2 wk after the final immunization. Data are the means of duplicate determinations. bullet, Control mice; {circ}, mice immunized with AdTSHR289; shaded circles, mice immunized with AdTSHR289 and antibody. N.D., Not done. C and D, Splenocytes were prepared 10 d after a single immunization with AdTSHR289 and/or anti-CD25 antibody and were stimulated with 5 µg/ml TSHR289 protein for 4 d. IFN{gamma} levels in the culture supernatants were measured by ELISA. Data are the mean ± SD of eight mice per group, expressed as picograms per milliliter.

 
In an in vitro recall assay, TSHR-specific IFN{gamma} production was comparable in splenocytes from both mouse strains immunized with AdTSHR289, which was not affected by CD4+CD25+ T cell depletion (Fig. 4Go, C and D). IL-4 was undetectable in all conditions (data not shown).

Lymphocyte infiltration in thyroid glands of hyperthyroid C57BL/6 mice
Thyroid histology, examined by H & E staining, demonstrated that regardless of CD4+CD25+ cell depletion, hyperthyroid BALB/c mice had diffuse goiters with hypertrophy and hypercellularity of thyroid epithelial cells, but no lymphocytic infiltration as previously reported (2, 3) (Fig. 5Go, D–F). However, the thyroid glands from two hyperthyroid C57BL/6 mice depleted of CD4+CD25+ T cells showed intrathyroidal lymphocytic infiltration and follicular destruction in addition to the features of hyperthyroidism (diffuse goiter and hypertrophy of thyroid epithelial cells; Fig. 5Go, G–I).


Figure 5
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  		FIG. 5. Thyroid histology. H & E-stained paraffin sections of thyroid glands from control BALB/c (A–C), hyperthyroid BALB/c (D–F), and hyperthyroid C57BL/6 (G–I) mice. Magnification: A, D, and G, x40; B, E, and H, x100; C, F, and I, x400.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We first show in this article that the resistance of C57BL/6 mice to AdTSHR289-induced Graves’ hyperthyroidism can be partly reversed by CD4+CD25+ T cell depletion. Thus, this immunological manipulation induced hyperthyroidism in 30% of C57BL/6 mice immunized with AdTSHR289 vs. 0% of those given AdTSHR289 alone. In addition, depletion of CD4+CD25+ T cells enhanced disease severity in susceptible BALB/c mice, as reflected by an approximately 2-fold increase in T4 levels in depleted mice compared with nondepleted mice. These results demonstrate a significant role for CD4+CD25+ T cells in disease susceptibility and severity in the adenovirus-TSHR-induced Graves’ hyperthyroidism model. In other words, CD4+CD25+ T cells play a role as suppressor cells in both susceptible and resistant mice. To our knowledge, this is the first study reporting the effect of CD4+CD25+ T cell depletion on autoimmune disease induction in susceptible and resistant mice in a single study.

Our data indicate that an imbalance between effector (pathogenic) and regulatory T cells may be one of the mechanisms involved in both disease development and severity in Graves’ hyperthyroidism. Thus, a difference in susceptibility to hyperthyroidism between two mouse strains cannot be attributed solely to the different regulatory effects of CD4+CD25+ T cells. In contrast, although reported separately, CD4+CD25+ T cell depletion converted otherwise resistant B10.S mice to susceptible to proteolipid protein-induced EAE (11), but had no effect on disease severity in EAE-susceptible SJL mice (16). Resistant B10.S mice had greater proportions of autoantigen-reactive CD4+CD25+ T cells than susceptible SJL mice (11), indicating the possible contribution of autoreactive CD4+CD25+ T cells to the resistance of B10.S mice to EAE. Similarly, using a thyroiditis model, Morris et al. (23) proposed that resistance to autoimmunity may, at least in some instances, reflect greater peripheral regulation, rather than a poor response to autoantigen. However, these researchers did not compare the effect of CD4+CD25+ T cell depletion between susceptible and resistant mice.

Recent data demonstrate that tolerance or resistance to EAE is due to either impaired function of APCs or T cell anergy (24, 25, 26). The resistance of C57BL/6 mice to AdTSHR289-induced hyperthyroidism is, however, unlikely to result from either of these mechanisms, because TSHR antibody production and the IFN{gamma} recall response in C57BL/6 mice were comparable with those in BALB/c mice, as shown in this article. However, we cannot exclude the possibility that the recall response of splenocytes to TSHR A-subunit antigen may be directed to clinically nonrelevant epitopes in resistant C57BL/6 mice, because we used a bulk of splenocytes. From our observations of TSHR antibody IgG subclasses and the splenocyte IFN-{gamma} recall response assay, immune imbalance of Th1 vs. Th2 is also an unlikely mechanism, although we recently demonstrated suppression of hyperthyroidism by immune deviation away from Th1 (22, 27). However, our present data, rather, indicate that CD4+CD25+ T cell depletion reversed resistance by tipping the balance of TSAb to TBAb toward TSAb dominance by enhancing TSAb production and/or decreasing TBAb synthesis. Shimojo et al. (28) and we (29) have previously demonstrated that differences in susceptibility to induction of hyperthyroidism among distinct mouse strains are largely attributed to nonmajor histocompatibility complex genetic background. Thus, the resistance defined by nonmajor histocompatibility complex genes could be partially overcome by CD4+CD25+ T cell depletion.

Numerical and/or functional impairments of CD4+CD25+ T cells have recently been demonstrated in several autoimmune diseases in both humans (30, 31, 32, 33, 34, 35) and mouse models (19, 36, 37), although the studies of the function of CD4+CD25+ T cells in autoimmune Hashimoto thyroiditis are controversial (30, 31). It will be of interest in the future to study the numbers and functional properties of CD4+CD25+ T cells in patients with Graves’ disease.

Of interest, unlike hyperthyroid BALB/c mice, thyroid glands from hyperthyroid C57BL/6 mice depleted of CD4+CD25+ T cells showed intrathyroidal lymphocytic infiltration and follicular destruction. However, it is unlikely that elevated T4 was due to thyroid destruction rather than thyroid stimulation by TSAb, because thyroid glands from these mice showed diffuse enlargement and cuboidal follicular epithelial cells. Thyroiditis has been reported in other Graves’ models, but inconsistent observations have been reported using the same immunization approach and within the same mouse strain (38). A recent study with thyroiditis-prone NOD.H-2h4 mice (39) showed no association of TSHR289-induced autoimmunity with thyroiditis. In particular, AdTSHR289 immunization induced Graves’ hyperthyroidism, but did not enhance thyroiditis or autoantibodies to thyroglobulin. Overall, these data indicate that in BALB/c and NOD.H-2h4 mice, immunization with AdTSHR289 induces antihuman TSHR antibodies that cross-react with mouse TSHR. In contrast, in C57BL/6 mice, induced immunity to the human TSHR can recruit mouse TSHR-reactive lymphocytes into the thyroid glands.

In conclusion, our CD4+CD25+ T cell depletion study demonstrates an important role for this T cell subpopulation in disease susceptibility and severity in a mouse Graves’ model, indicating that the imbalance between effector and regulatory T cells appears to be crucial in the pathogenesis of Graves’ disease.


   Acknowledgments
 
We thank Dr. K. Yui (Nagasaki University) for PC61 hybridoma. We also thank Drs. Sandra M. McLachlan and Basil Rapoport (Autoimmune Disease Unit, Cedars-Sinai Medical Center and University of California-Los Angeles) for providing us with AdTSHR289 and TSHR289 protein and for their helpful comments on the manuscript.


   Footnotes
 
The authors have no financial conflict of interest.

First Published Online January 26, 2006

Abbreviations: EAE, Experimental autoimmune encephalitis; H & E, hematoxylin and eosin; Th1, T helper 1; TBAb, thyroid-blocking antibody; TBIAb, TSH binding-inhibiting antibody; TSAb, thyroid-stimulating antibody; TSHR, TSH receptor.

Received August 11, 2005.

Accepted for publication January 18, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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O. Saitoh, N. Abiru, M. Nakahara, and Y. Nagayama
CD8+CD122+ T Cells, a Newly Identified Regulatory T Subset, Negatively Regulate Graves' Hyperthyroidism in a Murine Model
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