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Endocrinology Vol. 148, No. 12 6040-6046
Copyright © 2007 by The Endocrine Society

CD8+CD122+ T Cells, a Newly Identified Regulatory T Subset, Negatively Regulate Graves’ Hyperthyroidism in a Murine Model

Ohki Saitoh, Norio Abiru, Mami Nakahara and Yuji Nagayama

Department of Medical Gene Technology, Atomic Bomb Disease Institute (O.S., Y.N.), Division of Immunology, Endocrinology and Metabolism (N.A.), and Division of Clinical Pharmaceutics (M.N.), Department of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Graves’ disease is a thyroid-specific autoimmune disease mediated by stimulatory autoantibodies against the TSH receptor (TSHR). We have previously shown in our mouse model with adenovirus expressing the TSHR that antibody mediated depletion of CD4+CD25+ regulatory T cells (Tregs) enhances incidence and severity of hyperthyroidism in resistant and susceptible mouse strains, respectively. These data indicate that balance between effector T cells and Tregs is critical for disease development. This study was designed to evaluate the role played by another recently identified type of Treg, CD8+CD122+ T cells, in our mouse model to delineate the significance of different types of Tregs in Graves’ disease. Flow cytometry analysis showed that CD4+CD25+ and CD8+CD122+ T cells are distinct cell types, and anti-CD122 antibody effectively and selectively depleted CD8+CD122+ T cells. As for CD4+CD25+ Treg, CD8+CD122+ T cell depletion increased the incidence of hyperthyroidism both in resistant and susceptible mice. Of interest, intrathyroidal lymphocytic infiltration was observed in some CD8+CD122+ T cell-depleted, hyperthyroid resistant mice. These results indicate that in addition to CD4+CD25+ T cells, CD8+CD122+ T cells also play a crucial role in disease susceptibility in mouse Graves’ disease. Thus, different types of Tregs appear to be involved in tolerance to a self-antigen, the TSHR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GRAVES’ HYPERTHYROIDISM is a B-cell mediated, T-cell dependent autoimmune disease of the thyroid gland in which stimulatory autoantibodies against the TSH receptor (TSHR) [called thyroid stimulating antibody (TSAb)] cause overproduction of thyroid hormones and thyroid hyperplasia (1). The mechanisms for breakdown of immune tolerance to the TSHR remain unclear.

One concept for immune tolerance to "self" is that although autoreactive effector T cells are present in the periphery of virtually all individuals, they are kept in check by regulatory T cells (Tregs) (2). Thus, Tregs likely play a crucial role in peripheral tolerance of autoreactive T cells, which escape from thymic negative selection and migrate to the periphery. Tregs can be largely divided into two subpopulations: naturally occurring vs. inducible (3). CD4+CD25 (IL-2 receptor {alpha}-chain) + T cells are the most extensively studied naturally occurring Tregs, constitute 5–10% of peripheral CD4+ T cells, and are involved in the pathogenesis of a number of autoimmune diseases in both humans and mice (4). We have also previously reported in a mouse model of Graves’ disease with recombinant adenovirus expressing the TSHR that the incidence and severity of Graves’ hyperthyroidism are enhanced by depletion of CD4+CD25+ Treg with anti-CD25 monoclonal antibody (PC61) treatment in resistant C57BL/6 and susceptible BALB/c mice, respectively (5).

CD8+CD122 (IL-2 receptor ß chain) + T cells are another type of naturally occurring Tregs recently identified by Suzuki and colleagues (6, 7, 8). Mice genetically deficient for CD122 gene spontaneously develop severe hyperimmunity (6) by expansion of abnormally activated T cells (7). This abnormal phenotype can be reverted by transfer of purified CD8+CD122+ T cells (8). Of interest, a more recent study shows that there is a difference in the mechanism(s) of suppressive function between these two populations of Tregs, i.e. the suppressive function of CD4+CD25+ T cells is mediated by cell-cell contact (4), whereas that of CD8+CD122+ T cells is at least in part by secreting a regulatory cytokine IL-10, not by cell-cell contact (9).

Because we were interested in delineating the significance of different types of Tregs in Graves’ hyperthyroidism in our mouse model, this study was designed to study whether CD8+CD122+ T cells are also involved in the development of anti-TSHR autoimmunity and Graves’ hyperthyroidism as Tregs.


   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. Animal care and all experimental procedures were performed in accordance with the Guideline for Animal Experimentation of Nagasaki University with approval of the Institutional Animal Care and Use Committee.

Monoclonal antibodies
Anti-CD25 and anti-CD122 antibodies were purified from ascites of mice injected ip with hybridoma PC61 (5) or TMß-1 (a generous gift from Dr. T. Tanaka at Osaka University) (10, 11), respectively, with HiTrap protein G HP column (Amersham, Piscataway, NJ) according to the manufacturer’s instructions. In our preliminary data, the percentages of CD4+CD25+ T cells were declined from 3.22–0.77% by 0.5 mg anti-CD25 antibody and to 1.23% by 1 mg anti-CD25 antibody, and those of CD8+CD122+ T cells from 3.26–0.47% by 0.25 mg by anti-CD122 antibody and to 0.66% by 0.5 mg anti-CD122 antibodies. Therefore, 500 and 250 µg/mouse anti-CD25 and anti-CD122 antibodies, respectively, were used in the subsequent experiments.

Immunization protocols
Construction, amplification, and purification of nonreplicative recombinant human adenovirus expressing the human TSHR-A subunit (AdTSHR289; kindly provided by Drs. Sandra M. McLachlan and Basil Rapoport, Autoimmune Disease Unit, Cedars-Sinai Medical Center and University of California Los Angeles, CA), and determination of the viral particle concentration were described previously (5). Mice were injected im in the quadriceps with 100 µl PBS containing 1010 or 108 particles of AdTSHR289 (d 0) on three occasions at 3-wk intervals. Groups of mice were also treated by ip injection of 250 µg/mouse anti-CD122 monoclonal antibody on d 4 in each immunization. Blood, spleens, and thyroid tissues were obtained 2 wk after the final immunization.

Flow cytometry
Splenocytes were stained with fluorescein isothiocyanate or phycoerythrin-conjugated anti-CD4 (H129.19), anti-CD25 (7D4), anti-CD8 (53–6.7), and anti-CD122 (5H4) (PharMingen, San Diego, CA, or eBioscience, San Diego, CA), and analyzed on a FACScan flow cytometry using CellQuest software (BD Biosciences, Mountain View, CA). Note that the binding sites of 7D4 on CD25 and of 5H4 on CD122 are different from those of PC61 and TMß-1, respectively.

T4 and anti-TSHR antibody measurements
Serum free T4 concentrations were measured with a RIA kit (DPC free T4 kit; Diagnostic Products, Los Angeles, CA). The normal range was defined as the mean ± 3 SD of control untreated mice.

Anti-TSHR antibodies in mouse sera were determined using two different methods. First is a biological TSAb assay, which measures the stimulating antibodies responsible for hyperthyroidism and was performed with FRTL5 cells, as previously described (5). Briefly, the cells (5 x 104 cells per well in a 96-well culture plate) were incubated in 50 µl hypotonic Hanks’ balanced salt solution containing 0.5 mM isobutyl-methylxanthine, 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). Second is the flow cytometry assay with Chinese hamster ovary cells stably expressing the full-length TSHR (~2 x 106 receptors per cells), mouse sera (1:100 dilution), and fluorescein isothiocyanate-conjugated antimouse IgG antibody (Sigma-Aldrich Corp., St. Louis, MO), as previously described (12).

Cytokine assays
Splenocytes were cultured (triplicate aliquots) at 5 x 105 cells per well in a 96-well round-bottomed culture plate in the presence or absence of 5 µg/ml TSHR289 protein, as previously described (5). Four days later, the culture supernatants were collected. The concentrations of interferon (IFN)-{gamma}, IL-4, and IL-10 in these culture supernatants and mouse sera were determined with a Mouse IFN-{gamma} ELISA Set (PharMingen), a Mouse IL-4 ELISA Kit (BioSource Intl., Camarillo, CA), or a Mouse IL-10 ELISA Set (PharMingen), respectively. Cytokine production was expressed as nanogram or picogram per milliliter using a standard curve of recombinant mouse cytokine.

Thyroid histology
Thyroid histology was examined with two methods: hematoxylin and eosin (HE) staining of formalin-fixed tissue sections, and immunochemical staining of snap-frozen tissues sections with anti-CD4, anti-CD8 and anti-B220. In the latter, tissue sections were treated with 3% hydrogen peroxide to block endogenous peroxidase, reacted with primary antibodies [anti-CD4 (GK1.5) (1:2000 dilution), anti-CD8 (2.43) (1:4000 dilution), or anti-B220 (RA3-6B2) (1:4000 dilution)], and stained with a Histofine Simple Stain Mouse MAX-PO and diaminobenzidine solution (Nichirei, Tokyo, Japan).

Statistical analysis
Levels of T4, antibodies and cytokines, and incidences of hyperthyroidism were analyzed by a t or {chi}2 test, respectively. A P value less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of CD25 and CD122 expression on T-cell subpopulations and the efficacy of anti-CD25 and anti-CD122 antibody treatment
CD25 and CD122 are functionally closely related because these molecules are the {alpha} and ß-chains of the IL-2 receptor, respectively (13). Therefore, we first evaluated the expression pattern of these molecules on different T-cell subpopulations and the outcome of antibody mediated depletion using anti-CD25 or anti-CD122 antibodies. CD8+CD122+ T cells and CD4+CD25+ T cells constituted 2–4% of splenocytes (Fig. 1Go, A and D), as previously reported (4, 8). Splenocytes double positive for CD25 and CD122 or for CD8 and CD25 were negligible (Fig. 1Go, G and H). However, a small proportion (2–3%) of CD4+ T cells expressed low levels of CD122 (Fig. 1IGo and Table 1Go). Moderate amounts of CD4– or CD8– splenocytes also expressed low to high levels of CD122 (Fig. 1Go, D and I), which appear to be natural killer (NK) cells according to previous studies (10, 11).


Figure 1
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  		FIG. 1. Flow cytometric analysis of CD4, CD25, CD8, and CD122 expression on splenocytes. Mice were untreated or treated with 250 µg anti-CD122 antibody or 500 µg anti-CD25 antibody. Four days later, splenocytes were stained and analyzed by FACScan. The representative data with BLAB/c mice are shown. Similar results were also obtained with C57BL/6 mice. The squares a and b in I and J indicate CD122low and CD122high cells, respectively. Mice were left untreated (A, D, G, H, and I) or treated with anti-CD122 antibody (B, E, and J) or anti-CD25 antibody (C and F).

 

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  		TABLE 1. Percentages of T-cell subpopulations in splenocytes of control and antibody treated BALB/c mice1

 
Importantly, ip injection of anti-CD25 and anti-CD122 antibodies selectively depleted the corresponding T-cell subsets by 70–90%. In particular, mice injected with anti-CD122 antibody had a marked reduction in the proportion of CD8+CD122+ T cells (Fig. 1EGo) compared with the proportion of the same subset in untreated animals (Fig. 1DGo) or mice injected with anti-CD25 antibody (Fig. 1FGo). Likewise, the proportion of CD4+CD25+ T cells was reduced by anti-CD25 antibody (Fig. 1CGo) vs. the proportion of the same cell type in untreated animals (Fig. 1AGo) or mice injected with anti-CD122 antibody (Fig. 1BGo). Overall, these data clearly indicate that CD8+CD122+ T cells and CD4+CD25+ T cells are essentially distinct cell types and can be selectively depleted by either anti-CD25 or anti-CD122 antibodies, respectively. Regarding other CD122+ cells, because anti-CD122 antibody mainly depleted CD122high cells, although the reason(s) are unclear, the number of CD4+CD122low T cells was not affected, and approximately half the CD4–CD122+ or CD8–CD122+ NK cells was depleted (Fig. 1Go, E and J).

The effect of CD8+CD122+ T-cell depletion on anti-TSHR immune response and Graves’ hyperthyroidism in resistant C57BL/6 and susceptible BALB/c mice
Given the effective and selective depletion by anti-CD122 antibody of CD8+CD122+ T cells, we examined the role for CD8+CD122+ T cells in our mouse Graves’ model by antibody mediated depletion of this T-cell subset. Immunization with 1010 particles per mouse AdTSHR289 induced Graves’ hyperthyroidism in only four of 15 C57BL/6 mice (27%), consistent with our previous data showing that C57BL/6 mice are relatively resistant to immunization with adenovirus expressing the TSHR (14, 15). Depletion of CD8+CD122+ T cells significantly increased the disease induction rates up to 67% (10 of 15; P < 0.05 by {chi}2 test) (Fig. 2Go, A). However, in susceptible BALB/c mice, any effect of CD8+CD122+ T-cell depletion was obscured because 70% (seven of 10) of mice developed Graves’ hyperthyroidism by immunization with 1010 particles per mouse AdTSHR289 alone (Fig. 2BGo). Therefore, we immunized BALB/c mice with a lower dose of AdTSHR289 (108 particles per mouse, 100th the original amount). In this case, the incidence of Graves’ hyperthyroidism induced by AdTSHR289 alone was 30% (three of 10). These data are inconsistent with those in the previous report (16) showing high disease induction rates with a lower dose of AdTSHR289. The reason(s) for this difference is unclear, but subtle differences in immunization procedure and/or adenovirus purification may affect this difference. This incidence was increased to 70% (seven of 10) after antibody mediated CD8+CD122+ T-cell depletion (P < 0.05) (Fig. 2CGo). In addition, serum T4 levels were also significantly increased from 1.14 ± 0.63 ng/dl (mean ± SD) in nondepleted mice to 2.35 ± 0.94 ng/dl in depleted mice (P < 0.01). These results demonstrate that antibody mediated CD8+CD122+ T-cell depletion significantly enhances disease induction and/or severity of hyperthyroidism in resistant C57BL/6 and susceptible BALB/c mice.


Figure 2
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  		FIG. 2. Serum T4 concentrations in C57BL/6 (A) and BALB/c mice (B and C) immunized with 1010 (A and B) or 108 (C) particles per mouse AdTSHR289 without or after pretreatment with anti-CD122 antibody. T4 levels were determined 2 wk after the final immunization. Open and closed circles represent hyperthyroid and euthyroid mice, respectively. Horizontal lines designate normal upper limits of T4 values. *, A significant difference in disease incidence (P < 0.05). Control, Unimmunized mice.

 
Anti-TSHR antibodies were determined using two different methods. The TSAb assay measures the stimulating antibodies responsible for hyperthyroidism. The flow cytometry assay using Chinese hamster ovary cells expressing the full-length TSHR detects anti-TSHR antibodies, regardless of their function. There were no statistically significant differences in antibody titers determined with the two assays between the immunized mice with/without CD8+CD122+ T-cell depletion. This may be at least partly due to wide variations of antibody titers. However, TSAb activities and anti-TSHR antibody titers tended to be higher in mice depleted of CD8+CD122+ T cells compared with nondepleted mice in both mouse strains (Fig. 3Go), and there was a weak but significant correlation between serum T4 levels and TSAb titers (r = 0.39; P < 0.05) (Fig. 4Go). Similar weak but significant correlations between T4 and TSAb were previously observed in our first Graves’ model with AdTSHR (14) and also in our other Graves’ models with dendritic cells infected with AdTSHR or AdTSHR289 (12, 17). This is at least partly due to the fact that the net effect of TSAb and blocking antibody, not TSAb by itself, determines thyroid function levels.


Figure 3
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  		FIG. 3. TSAb (A and B) and anti-TSHR antibody values (C and D) in C57BL/6 (A and C) and BALB/c mice (B and D) immunized with AdTSHR289 without or after pretreatment with anti-CD122 antibody. Sera from mice in A and C of Fig. 2Go were used. Open and closed circles represent hyperthyroid and euthyroid mice.

 

Figure 4
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  		FIG. 4. Relationship between T4 and TSAb. The data on mice immunized with AdTSHR289 with/without antibodies were used. Correlation coefficient is 0.39 (P < 0.05). Open and closed circles designate C57BL/6 and BALB/c, respectively.

 
Antigen-specific in vitro cytokine secretion from splenocytes was also examined. In our previous study (19, 20), splenocytes from mice immunized with adenovirus expressing the full-length TSHR exhibited antigen-specific IFN-{gamma} and, to a lesser extent, IL-10, but not IL-4, secretion. TSHR-specific IFN-{gamma} production was significantly higher in splenocytes from CD8+CD122+ T-cell depleted C57BL/6 mice than those from nondepleted mice (23.2 ± 10.7 vs. 8.2 ± 2.9 ng/ml, mean ± SD; P < 0.01) (Fig. 5Go, A). However, no difference was observed in BALB/c mice (Fig. 5BGo). TSHR-specific IL-10 production was minimum in immunized C57BL/6 and BALB/c mice (IL-10 production was less with adenovirus expressing the TSHR-A subunit compared with that with adenovirus coding the full-length TSHR in our previous study) (20), and a significant increase in TSHR-specific IL-10 production was observed only in CD8+CD122+ T-cell depleted C57BL/6 mice (Fig. 5Go, C and D). TSHR-specific IL-4 production was not observed in all the mice (data not shown). Furthermore, serum levels of these cytokines were undetectable in all the mice.


Figure 5
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  		FIG. 5. IFN-{gamma} and IL-10 production from splenocytes of mice treated with AdTSHR289 alone and in combination with anti-CD122 antibody. Spleen from mice in A and C of Fig. 2Go were used. Splenocytes were stimulated with 5 µg/ml TSHR289 protein for 4 d. IFN-{gamma} and IL-10 levels in the culture supernatants were measured by ELISA. Data are expressed as mean ± SD of four mice per group. Open and closed bars represent splenocytes cultured in the absence or presence of TSHR289. * and **, A significant difference (P < 0.05 and < 0.01, respectively).

 
Thyroid histology in mice depleted of CD8+CD122+ T cells
In histological examination of the thyroid glands by HE staining, the thyroid glands from three C57BL/6 mice treated with AdTSHR289 and anti-CD122 antibody, but none of C57BL/6 mice treated with AdTSHR289 alone, showed intrathyroidal lymphocytic infiltration (Fig. 6CGo). Interestingly, all of these mice with intrathyroidal lymphocytic infiltration were hyperthyroid. In contrast, no intrathyroidal lymphocytic infiltration was seen in BALB/c mice treated with AdTSHR289 and anti-CD122 antibody (Fig. 6BGo). These data are consistent with our previous report using anti-CD25 antibody (5). Immunohistochemical analysis of these specimens (n = 2) revealed that the infiltrated lymphocytes largely constitute CD4+ T cells, not CD8+ T cells or B cells (Fig. 6Go, D and E).


Figure 6
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  		FIG. 6. Thyroid histology. HE staining of the thyroid glands from control C57BL/6 (A), hyperthyroid BALB/c (B), and hyperthyroid C57BL/6 mice with lymphocyte infiltration (C). Immunohistochemical (IHC) analysis of the thyroid glands from hyperthyroid C57BL/6 mice (D, control; E, stained with anti-CD4 antibody). Magnifications, x100 (A, B, and C) and x400 (D and E).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The receptor for cytokine IL-2 is composed of three subunits, called {alpha} (CD25), ß (CD122), and {gamma} (CD132) (13). In mice, the {alpha}-chain is required for binding to IL-2 (21). Whereas the {gamma}-chain is common to a family of cytokine receptors, including IL-2, 4, 7, 13, and 15, and is constitutively expressed on all the lymphocyte subsets, the expression of the {alpha} and ß-chains is restricted to particular subsets of immune cells. Thus, Tanaka et al. (10) have reported that the expression of CD122 is confined to CD8+ T cells and NK1.1+ NK cells, and is negligible on CD4+ T cells in spleen of C57BL/6 mice. Suzuki and colleagues (8) have also recently demonstrated that peripheral CD4+CD25+ T cells lack expression of CD122 in C3H/He mice. In contrast, low levels of CD122 expression have been reported on CD4+CD25+ Tregs (22), particularly on "activated" Tregs (23). Our study clearly shows that CD4+CD25+ T cells and CD8+CD122+ T cells do not express CD122 or CD25, respectively, and that treatment with anti-CD122 antibody (or anti-CD25 antibody) does not affect the percentage of CD4+CD25+ T cells (or CD8+CD122+ T cells). Although the reason(s) for the discrepant data between our data and previous observations (22, 23) are unknown, our findings clearly demonstrate that each antibody can be used to deplete selectively a respective T-cell subset.

Having confirmed the efficient and selective depletion of CD8+CD122+ T cells by anti-CD122 antibody, we evaluated the role for this newly identified subset of Tregs, CD8+CD122+ T cells, by antibody mediated depletion in the pathogenesis of Graves’ hyperthyroidism in our mouse model. Here, we found enhancement of Graves’ hyperthyroidism by depletion of this Treg subset, however: 1) the suppressive effect of CD8+CD122+ Treg depletion on BALB/c mice immunized with a lower, but not a higher, dose of adenovirus; and 2) enhanced splenocyte secretions of IFN-{gamma} and IL-10 in depleted C57BL/6, but not BALB/c, mice apparently suggest a limited impact of CD8+CD122+ Tregs on susceptible BALB/c, compared with resistant C57BL/6 mice. These data may raise a possibility for a role of CD8+CD122+ Tregs in nonsusceptibility of C57BL/6 mice. However, another possibility that anti-TSHR immune response may already be fully induced by only adenovirus immunization in BALB/c mice cannot be completely excluded, which likely makes the effect of Tregs obscure in this susceptible mouse.

These results, together with our previous study (5), demonstrate that both CD4+CD25+ and CD8+CD122+ Tregs negatively regulate experimentally induced Graves’ disease. The fact that depletion of either Treg exacerbated Graves’ hyperthyroidism suggests that suppressive mechanism(s) of each Treg may not be identical. Indeed, there are some differences in their functional properties, as described in the beginning of this article. Nevertheless, we found no additive effect of anti-CD122 and anti-CD25 antibodies in our mouse model (data not shown). In addition, the significant enhanced effect of CD4+CD25+ Treg depletion in BALB/c mice immunized with a higher dose of adenovirus in our previous study (5) indicates the predominant effect of CD4+CD25+ Tregs over CD8+CD122+ Tregs.

Furthermore, a possibility that depletion of CD8–CD122+ NK cells contributes to these results also cannot completely be excluded but is very unlikely because hyperthyroidism is caused by humoral immune response (e.g. anti-TSHR antibody), not by cellular immune response such as cytotoxic T cells and NK cells.

Interestingly, both CD4+CD25+ Treg and CD8+CD122+ Treg depletion induced not only hyperthyroidism, but also intrathyroidal lymphocyte infiltration in some hyperthyroid C57BL/6 mice. Intrathyroidal lymphocyte infiltration is observed to some extent in the thyroid glands from Graves’ patients but is also a hallmark of Hashimoto thyroiditis. Although it is unclear whether intrathyroidal lymphocyte infiltration in Graves’ patients involves anti-TSHR immune response or immune response to other thyroid autoantigens such as thyroid peroxidase and thyroglobulin (Tg), the positive correlation between the degree of lymphocyte infiltration and anti-thyroid peroxidase/Tg antibody titers (24, 25) supports the latter possibility. However, our data indicate that immunization with AdTSHR289 can induce cellular immune response to the TSHR in certain circumstances. It is unknown why intrathyroidal lymphocyte infiltration can be induced only in Graves’ disease-resistant C57BL/6 mice treated with antibodies to Tregs, CD4+CD25+ or CD8+CD122+ T cells.

In mouse models of Hashimoto thyroiditis, anti-CD25 antibody has recently been reported to overcome the resistance of Tg-tolerized CBA/J mice and of BALB/c mice to Tg-induced thyroiditis (26, 27). Suppression of Tg-induced experimental thyroiditis by CD4+CD25+ Tregs induced by granulocyte macrophage-colony stimulating factor or by immature dendritic cells has also been demonstrated (28, 29). Although lack of the effect of anti-CD25 antibody has recently been reported in nonobese diabetic (NOD-H2h4) mice of a spontaneous thyroiditis model, in which mice were given three weekly injections of anti-CD25 antibody beginning 10–11 d after birth (30), our recent study showed that injection of anti-CD25 antibody starting 4 d before the initiation of iodine in the drinking water significantly exacerbates the degree of lymphocyte infiltration into the thyroid glands in NOD-H2h4 mice (31). Thus, CD4+CD25+ Tregs appear to play an essential role in the pathogenesis of not only Graves’ disease but also Hashimoto thyroiditis in animal models. However, antibody mediated depletion of CD8+CD122+ T cells did not influence the incidence of thyroiditis in NOD-H2h4 mice (our unpublished data). It is at present unknown whether these data indicate insignificance of CD8+CD122+ cells or significance of NK cells in the pathogenesis of thyroiditis.

There is no definitive evidence showing that autoimmune thyroid disease results from numerical and/or functional abnormalities of Tregs in humans. A recent study by Marazuela et al. (32) has demonstrated that a number of CD4+CD25+ T cells increases in thyroid glands from Graves’ patients with impaired suppressor functions. It remains to be investigated whether suppressor function of Tregs is intrinsically impaired in Graves’ patients or whether the thyroid microenvironments induce this abnormality. In Hashimoto thyroiditis in humans, one study has shown a reduced suppressive capacity of CD4+CD25+ Tregs (33), but another has not (34). Further investigation will be required to dissect the significance of Tregs in human autoimmune thyroid diseases.

Recent studies have also reported different types of naturally occurring Tregs in a CD8+ T-cell lineage (18, 35, 36). Similarity and dissimilarity of these CD8+ Tregs remain unclear, but it is likely that numerous distinct Tregs of different lineages (CD4+ vs. CD8+; naturally occurring vs. inducible) intertwine intricately in the immune network system. Although there are still numerous issues of Tregs to be solved in mice and humans, hopefully, in the not-so distant future, manipulation of Tregs may provide novel ways to treat immunological disorders.


   Acknowledgments
 
We thank Dr. K. Yui at Nagasaki University for hybridomas PC61. We also thank Drs. Sandra M. McLachlan and Basil Rapoport, Autoimmune Disease Unit, Cedars-Sinai Medical Center and University of California Los Angeles, California, for critical reading of the manuscript.


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online September 6, 2007

Abbreviations: HE, Hematoxylin and eosin; IFN, interferon; NK, natural killer; NOD-H2h4, nonobese diabetic; Tg, thyroglobulin; Treg, regulatory T cell; TSAb, thyroid stimulating antibody; TSHR, TSH receptor.

Received March 5, 2007.

Accepted for publication August 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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