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.

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SummaryMajor histocompatibility complex (MHC) class I-restricted T cell epitopes are generated mainly by the immunoproteasome in antigen-presenting cells. Therefore, inhibition of activity of this proteolytic complex molecule is thought to be a potential treatment for cell-mediated autoimmune diseases. We therefore studied the efficacy of an immunoproteasome inhibitor, ONX 0914 (formerly PR-957), for the treatment of autoimmune thyroid diseases, including cell-mediated Hashimoto's thyroiditis and autoantibody-mediated Graves' hyperthyroidism using mouse models. Our data show that ONX 0914 was effective prophylactically and therapeutically at suppressing the degree of intrathyroidal lymphocyte infiltration and, to a lesser degree, the titres of anti-thyroglobulin autoantibodies in non-obese diabetic (NOD)-H2 h4 mice, an iodine-induced autoimmune thyroiditis model. It also inhibited differentiation of T cells to T helper type 1 (Th1) and Th17 cells, effector T cell subsets critical for development of thyroiditis in this mouse strain. In contrast, its effect on the Graves' model was negligible. Although ONX 0914 exerts its immune-suppressive effect through not only suppression of immune proteasome but also other mechanism(s), such as inhibition of T cell differentiation, the present results suggest that the immunoproteasome is a novel drug target in treatment of Hashimoto's thyroiditis in particular and cell-mediated autoimmune diseases in general.

The mutant BRAF (BRAF(V600E)) is the most common genetic alteration in papillary thyroid carcinomas (PTCs). The oncogenicity of this mutation has been shown by some genetically engineered mouse models. However, in these mice, BRAF(V600E) is expressed in all the thyroid cells from the fetal periods, and suppresses thyroid function, thereby leading to TSH elevation, which by itself promotes thyroid tumorigenesis. To overcome these problems, we exploited 2 different approaches, both of which allowed temporally and spatially restricted expression of BRAF(V600E) in the thyroid glands. First, we generated conditional transgenic mice harboring the loxP-neo(R)-loxP-BRAF(V600E)-internal ribosome entry site-green fluorescent protein sequence [Tg(LNL-BRAF(V600E))]. The double transgenic mice (LNL-BRAF(V600E);TPO-Cre) were derived from a high expressor line of Tg(LNL-BRAF(V600E)) mice and TPO-Cre mice; the latter expresses Cre DNA recombinase under the control of thyroid-specific thyroid peroxidase (TPO) promoter and developed PTC-like lesions in early life under normal serum TSH levels due to mosaic recombination. In contrast, injection of adenovirus expressing Cre under the control of another thyroid-specific thyroglobulin (Tg) promoter (Ad-TgP-Cre) into the thyroids of LNL-BRAF(V600E) mice did not induce tumor formation despite detection of BRAF(V600E) and pERK in a small fraction of thyroid cells. Second, postnatal expression of BRAF(V600E) in a small number of thyroid cells was also achieved by injecting the lentivirus expressing loxP-green fluorescent protein-loxP-BRAF(V600E) into the thyroids of TPO-Cre mice; however, no tumor development was again observed. These results suggest that BRAF(V600E) does not appear to induce PTC-like lesions when expressed in a fraction of thyroid cells postnatally under normal TSH concentrations.

Graves-like hyperthyroidism is induced in BALB/c mice by immunization with adenovirus expressing the human TSH receptor (TSHR) A-subunit (amino acids 1-289). However, because of nonidentity between the human and mouse TSHR ( approximately 87% amino acid homology), we compared the responses of mice immunized with adenoviruses expressing either the mouse or the human TSHR A-subunit. Wild-type (wt) BALB/c mice immunized with the mouse A-subunit developed neither TSHR antibodies (measured by flow cytometry) nor thyroid lymphocytic infiltration. However, wt C57BL/6 mice developed sparse intrathyroidal lymphocyte infiltration without antibody production. Depletion of naturally occurring regulatory CD4(+)CD25(+) T cells had little effect. These results indicate the inability to break tolerance to the mouse TSHR in wt mice. In contrast, TSHR knockout (KO) BALB/c mice generated mouse TSHR antibodies in response to mouse A-subunit immunization and augmented human TSHR antibody response to human A-subunit immunization. Thyroid-stimulating antibody titers measured in a functional bioassay were comparable in human A-subunit immunized wt mice and in TSHR KO mice immunized with either the mouse or human A-subunit. In conclusion, immune response to the mouse TSHR is readily induced in TSHR KO but not in wt mice. Only in the former does immunization with adenovirus expressing the mouse A-subunit generate antibodies capable of activating the mouse TSHR. TSHR KO mice are, therefore, of value for future studies dissecting the autoimmune response to the mouse TSHR.

We have recently shown that wild type mice are highly tolerant, whereas thyrotropin receptor (TSHR) knockout (KO) mice are susceptible to immunization with the mouse TSHR, the autoantigen in Graves' disease. However, because TSHR KO mice lack the endogenous TSHR, Graves-like hyperthyroidism cannot be expected to occur in these mice. We therefore performed adoptive transfer of splenocytes from TSHR KO mice into nude mice expressing the endogenous TSHR. Anti-TSHR autoantibodies were detected in approximately 50 % recipient mice 4 wk after adoptive transfer of splenocytes (5 × 10⁷/mouse) from TSHR KO mice immunized with adenovirus expressing mTSHR A subunit and persisted for 24 wk. Depletion of regulatory T cells by anti-CD25 antibody in the donor mice increased successful transfer rates without increasing antibody levels. Some recipient mice showed transient increases in thyroid-stimulating antibodies and T₄ levels 4-8 wk after transfer, but many became thyroid-blocking antibody positive and hypothyroid 24 wk later. Adoptive transfer of splenocytes from naïve TSHR KO mice transiently induced very low antibody titers when the recipient mice were treated with anticytotoxic lymphocyte antigen 4 and antiprogrammed cell death 1 ligand 1 antibodies for 8 wk after transfer. Histologically, macrophages infiltrated the retrobulbar adipose tissues and extraocular muscles in a small fraction of the recipients. Our findings demonstrate successful adoptive transfer of anti-TSHR immune response from TSHR KO mice to nude mice. Although the recipient mice developed only transient and infrequent hyperthyroidism, followed by eventual hypothyroidism, induction of orbital inflammation suggests the possible role of anti-TSHR immune response for Graves' orbitopathy.

SummaryGraves' disease is a B cell-mediated and T cell-dependent autoimmune disease of the thyroid which is characterized by overproduction of thyroid hormones and thyroid enlargement by agonistic anti-thyrotrophin receptor (TSHR) autoantibody. In addition to antibody secretion, B cells have recently been recognized to function as antigen-presenting/immune-modulatory cells. The present study was designed to evaluate the efficacy of B cell depletion by anti-mouse (m) CD20 monoclonal antibody (mAb) on Graves' hyperthyroidism in a mouse model involving repeated injection of adenovirus expressing TSHR A-subunit (Ad-TSHR289). We observe that a single injection of 250 mg/ mouse anti-mCD20 mAb eliminated B cells efficiently from the periphery and spleen and to a lesser extent from the peritoneum for more than 3 weeks. B cell depletion before immunization suppressed an increase in serum immunoglobulin (Ig)G levels, TSHR-specific splenocyte secretion of interferon (IFN)-g, anti-TSHR antibody production and development of hyperthyroidism. B cell depletion 2 weeks after the first immunization, a time-point at which T cells were primed but antibody production was not observed, was still effective at inhibiting antibody production and disease development without inhibiting splenocyte secretion of IFN-g. By contrast, B cell depletion in hyperthyroid mice was therapeutically ineffective. Together, these data demonstrate that B cells are critical not only as antibody-producing cells but also as antigenpresenting/immune-modulatory cells in the early phase of the induction of experimental Graves' hyperthyroidism and, although therapeutically less effective, B cell depletion is highly efficient for preventing disease development.

The non-obese diabetic (NOD) mouse spontaneously develops several autoimmune diseases, including type 1 diabetes and to a lesser extent thyroiditis and sialitis. Imbalance between effector T cells (Teffs) and regulatory T cells (Tregs) has recently been proposed as a mechanism for the disease pathogenesis in NOD mice, but previous studies have shown the various outcomes by different timing and methods of Treg-depletion. This study was therefore designed to compare the consequences of Treg-depletion by the same method (anti-CD25 antibody) on the spectrum of organ-specific autoimmune diseases in NOD mice of different ages. Treg-depletion by anti-CD25 antibody at 10 days of age accelerated development of all three diseases we examined (insulitis/diabetes, thyroiditis and sialitis); Treg-depletion at 4 weeks of age accelerated only diabetes but not thyroiditis or sialitis; and Treg-depletion at 12 weeks of age hastened only development of thyroiditis and exhibited little influence on diabetes or sialitis. Increased levels of insulin autoantibodies (IAA) were however observed in mice depleted of Tregs at 10 days of age, not in those at 4 weeks. Thus, the consequences of Treg-depletion on the spectrum of organ-specific autoimmune diseases depend on the timing of anti-CD25 antibody injection in NOD mice. Aging gradually tips balance between Teffs and Tregs toward Teff-dominance for diabetes, but this balance for thyroiditis and sialitis likely alters more intricately. Our data also suggest that the levels of IAA are not necessarily correlated with diabetes development.