Requirement for CD40/CD40L Interactions for Development of Autoimmunity Differs Depending on Specific Checkpoint and Costimulatory Pathways.

CD40/CD40L interactions play a critical role in immunity and autoimmunity. In this study, we sought to understand the requirement for CD40 signaling in the programmed cell death-1 (PD-1) checkpoint and CD28 costimulatory pathways important for maintenance of peripheral tolerance. Blocking either pathway can result in loss of self-tolerance and development of autoimmunity. We found that primary Sjögren's syndrome (pSS) and autoimmune thyroid diseases (ATDs) that develop spontaneously in CD28-deficient IFN-γ-/- NOD.H-2h4 (CD28-/-) mice required CD40 signaling. Specifically, blockade of CD40L with the anti-CD40L mAb, MR1, inhibited autoantibody production and inflammation in thyroid and salivary gland target tissues. Unexpectedly, however, ATD and pSS in PD-1-deficient IFN-γ-/- NOD.H-2h4 (PD-1-/-) mice developed independently of CD40/CD40L interactions. Treatment with MR1 had no effect and even exacerbated disease development in pSS and ATD, respectively. Most interesting, anti-thyroglobulin and pSS-associated autoantibodies were increased following anti-CD40L treatment, even though MR1 effectively inhibited the spontaneous splenic germinal centers that form in PD-1-deficient mice. Importantly, blockade of the PD-1 pathway by administration of anti-PD-1 mAb in CD28-/- mice recapitulated the PD-1-/- phenotype, significantly impacting the ability of MR1 to suppress ATD and pSS in these mice. These results indicate that there can be different pathways and requirements to autoimmune pathogenesis depending on the availability of specific checkpoint and costimulatory receptors, and an intact PD-1 pathway is apparently required for inhibition of autoimmunity by anti-CD40L.

Mechanisms by Which B Cells and Regulatory T Cells Influence Development of Murine Organ-Specific Autoimmune Diseases.

Experiments with B cell-deficient (B-/-) mice indicate that a number of autoimmune diseases require B cells in addition to T cells for their development. Using B-/- Non-obese diabetic (NOD) and NOD.H-2h4 mice, we demonstrated that development of spontaneous autoimmune thyroiditis (SAT), Sjogren's syndrome and diabetes do not develop in B-/- mice, whereas all three diseases develop in B cell-positive wild-type (WT) mice. B cells are required early in life, since reconstitution of adult mice with B cells or autoantibodies did not restore their ability to develop disease. B cells function as important antigen presenting cells (APC) to initiate activation of autoreactive CD4+ effector T cells. If B cells are absent or greatly reduced in number, other APC will present the antigen, such that Treg are preferentially activated and effector T cells are not activated. In these situations, B-/- or B cell-depleted mice develop the autoimmune disease when T regulatory cells (Treg) are transiently depleted. This review focuses on how B cells influence Treg activation and function, and briefly considers factors that influence the effectiveness of B cell depletion for treatment of autoimmune diseases.

Autoimmune manifestations in aged mice arise from early-life immune dysregulation.

Autoantibodies can be present years to decades before the onset of disease manifestations in autoimmunity. This finding suggests that the initial autoimmune trigger involves a peripheral lymphoid component, which ultimately drives disease pathology in local tissues later in life. We show that Sjögren's syndrome manifestations that develop in aged NOD.H-2h4 mice were driven by and dependent on peripheral dysregulation that arose in early life. Specifically, elimination of spontaneous germinal centers in spleens of young NOD.H-2h4 mice by transient blockade of CD40 ligand (CD40L) or splenectomy abolished Sjögren's pathology of aged mice. Strikingly, a single injection of anti-CD40L at 4 weeks of age prevented tertiary follicle neogenesis and greatly blunted the formation of key autoantibodies implicated in glandular pathology, including anti-muscarinic receptor antibodies. Microarray profiling of the salivary gland characterized the expression pattern of genes that increased with disease progression and showed that early anti-CD40L greatly repressed B cell function while having a broader effect on multiple biological pathways, including interleukin-12 and interferon signaling. A single prophylactic treatment with anti-CD40L also inhibited the development of autoimmune thyroiditis and diabetes in NOD.H-2h4 and nonobese diabetic mice, respectively, supporting a key role for CD40L in the pathophysiology of several autoimmune models. These results strongly suggest that early peripheral immune dysregulation gives rise to autoimmune manifestations later in life, and for diseases predated by autoantibodies, early prophylactic intervention with biologics may prove efficacious.

New Murine Model of Early Onset Autoimmune Thyroid Disease/Hypothyroidism and Autoimmune Exocrinopathy of the Salivary Gland.

Sixty to seventy percent of IFN-γ(-/-) NOD.H-2h4 mice given sodium iodide (NaI)-supplemented water develop a slow onset autoimmune thyroid disease, characterized by thyrocyte epithelial cell (TEC) hyperplasia and proliferation (H/P). TEC H/P develops much earlier in CD28(-/-) mice and nearly 100% (both sexes) have severe TEC H/P at 4 mo of age. Without NaI supplementation, 50% of 5- to 6-mo-old CD28(-/-)IFN-γ(-/-) mice develop severe TEC H/P, and 2-3 wk of NaI is sufficient for optimal development of severe TEC H/P. Mice with severe TEC H/P are hypothyroid, and normalization of serum thyroxine levels does not reduce TEC H/P. Activated CD4(+) T cells are sufficient to transfer TEC H/P to SCID recipients. Thyroids of mice with TEC H/P have infiltrating T cells and expanded numbers of proliferating thyrocytes that highly express CD40. CD40 facilitates, but is not required for, development of severe TEC H/P, as CD40(-/-)IFN-γ(-/-)CD28(-/-) mice develop severe TEC H/P. Accelerated development of TEC H/P in IFN-γ(-/-)CD28(-/-) mice is a result of reduced regulatory T cell (Treg) numbers, as CD28(-/-) mice have significantly fewer Tregs, and transfer of CD28(+) Tregs inhibits TEC H/P. Essentially all female IFN-γ(-/-)CD28(-/-) NOD.H-2h4 mice have substantial lymphocytic infiltration of salivary glands and reduced salivary flow by 6 mo of age, thereby providing an excellent new model of autoimmune exocrinopathy of the salivary gland. This is one of very few models where autoimmune thyroid disease and hypothyroidism develop in most mice by 4 mo of age. This model will be useful for studying the effects of hypothyroidism on multiple organ systems.

Mechanisms and kinetics of proliferation and fibrosis development in a mouse model of thyrocyte hyperplasia.

IFN-γ(-/-) NOD.H-2h4 mice develop autoimmune disease with extensive hyperplasia and proliferation of thyroid epithelial cells (TEC H/P) and fibrosis. Splenic T cells from donors with severe TEC H/P transfer TEC H/P to SCID recipients. The goal of this study was to determine what factors control TEC H/P development/progression by examining T cells, markers of apoptosis, senescence and proliferation in thyroids of SCID recipients over time. At 28days, T cell infiltration was maximal, thyrocytes were proliferating, and fibrosis was moderate. At days 60 and 90, thyroids were larger with more fibrosis. T cells, cytokines and thyrocyte proliferation decreased, and cell cycle inhibitor proteins, and anti-apoptotic molecules increased. T cells and thyrocytes had foci of phosphorylated histone protein H2A.X, indicative of cellular senescence, when TEC H/P progressed and thyrocyte proliferation declined. Some thyrocytes were regenerating at day 90, with irregularly shaped empty follicles and ciliated epithelium. Proliferating thyrocytes were thyroid transcription factor (TTF1)-positive, suggesting they derived from epithelial cells and not brachial cleft remnants.

NOD.H-2h4 mice: an important and underutilized animal model of autoimmune thyroiditis and Sjogren's syndrome.

NOD.H-2h4 mice express the K haplotype on the NOD genetic background. They spontaneously develop thyroiditis and Sjogren's syndrome, but they do not develop diabetes. Although autoimmune thyroid diseases and Sjogren's syndrome are highly prevalent autoimmune diseases in humans, there has been relatively little emphasis on the use of animal models of these diseases for understanding basic mechanisms involved in development and therapy of chronic organ-specific autoimmune diseases. The goal of this review is to highlight some of the advantages of NOD.H-2h4 mice for studying basic mechanisms involved in development of autoimmunity. NOD.H-2h4 mice are one of relatively few animal models that develop organ-specific autoimmune diseases spontaneously, i.e., without a requirement for immunization with antigen and adjuvant, and in both sexes in a relatively short period of time. Thyroiditis and Sjogren's syndrome in NOD.H-2h4 mice are chronic autoimmune diseases that develop relatively early in life and persist for the life of the animal. Because the animals do not become clinically ill, the NOD.H-2h4 mouse provides an excellent model to test therapeutic protocols over a long period of time. The availability of several mutant mice on this background provides a means to address the impact of particular cells and molecules on the autoimmune diseases. Moreover, to our knowledge, this is the only animal model in which the presence or absence of a single cytokine, IFN-γ, is sufficient to completely inhibit one autoimmune thyroid disease, with a completely distinct autoimmune thyroid disease developing when it is absent.

Regulatory T cells in B-cell-deficient and wild-type mice differ functionally and in expression of cell surface markers.

NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) with chronic inflammation of thyroids by T and B cells. B-cell deficient (B(-/-) ) mice are resistant to SAT but develop SAT if regulatory T (Treg) cells are transiently depleted. We established a transfer model using splenocytes from CD28(-/-)  B(-/-) mice (effector cells and antigen-presenting cells) cultured with or without sorted Treg cells from Foxp3-GFP wild-type (WT) or B(-/-) mice. After transfer to mice lacking T cells, mice given Treg cells from B(-/-) mice had significantly lower SAT severity scores than mice given Treg cells from WT mice, indicating that Treg cells in B(-/-) mice are more effective suppressors of SAT than Treg cells in WT mice. Treg cells from B(-/-) mice differ from WT Treg cells in expression of CD27, tumour necrosis factor receptor (TNFR) II p75, and glucocorticoid-induced TNFR-related protein (GITR). After transient depletion using anti-CD25 or diphtheria toxin, the repopulating Treg cells in B(-/-) mice lack suppressor function, and expression of CD27, GITR and p75 is like that of WT Treg cells. If B(-/-) Treg cells develop with B cells in bone marrow chimeras, their phenotype is like that of WT Treg cells. Addition of B cells to cultures of B(-/-) Treg and T effector cells abrogates their suppressive function and their phenotype is like that of WT Treg cells. These results establish for the first time that Treg cells in WT and B(-/-) mice differ both functionally and in expression of particular cell surface markers. Both properties are altered after transient depletion and repopulation of B(-/-) Treg cells, and by the presence of B cells during Treg cell development or during interaction with effector T cells.

Follicular B cells in thyroids of mice with spontaneous autoimmune thyroiditis contribute to disease pathogenesis and are targets of anti-CD20 antibody therapy.

B cells are required for development of spontaneous autoimmune thyroiditis (SAT) in NOD.H-2h4 mice where they function as important APCs for activation of CD4(+) T cells. Depletion of B cells using anti-CD20 effectively inhibits SAT development. The goals of this study were to characterize the B cells that migrate to thyroids in SAT, and to determine whether anti-CD20 effectively targets those B cells in mice with established SAT. The results showed that most thyroid-infiltrating B cells in mice with SAT are follicular (FO) B cells. Expression of CD80, CD86, and CD40 was significantly increased on FO, but not marginal zone, splenic B cells after SAT development. Thyroid-infiltrating and peripheral blood B cells had lower expresion of CD20 and CD24 compared with splenic and lymph node FO B cells. Despite reduced CD20 expression, anti-CD20 depleted most B cells in thyroids of mice with established SAT within 3 d. B cell depletion in thyroids of mice given anti-CD20 was more complete and longer lasting than in spleen and lymph nodes and was comparable to that in blood. Circulation of B cells was required for effective and rapid removal of B cells in thyroids because preventing lymphocyte egress by administration of FTY720 abrogated the effects of anti-CD20 on thyroid B cells. Therefore, the FO subset of B cells preferentially contributes to SAT development and persistence, and anti-CD20 targeting of FO B cells effectively eliminates B cells in the target organ even though thyroid B cells have decreased CD20 expression.

Culture promotes transfer of thyroid epithelial cell hyperplasia and proliferation by reducing regulatory T cell numbers.

IFN-γ(-/-) NOD.H-2h4 mice develop a spontaneous autoimmune thyroid disease, thyroid epithelial cell hyperplasia and proliferation (TEC H/P) when given NaI in their water for 7+ mo. TEC H/P can be transferred to IFN-γ(-/-) SCID mice by splenocytes from mice with severe (4-5+) disease, and transfer of TEC H/P is improved when splenocytes are cultured prior to transfer. Older (9+ mo) IFN-γ(-/-) NOD.H-2h4 mice have elevated numbers of FoxP3(+) T reg cells, up to 2-fold greater than younger (2 mo) mice. During culture, the number of T reg decreases and this allows the improved transfer of TEC H/P. Co-culture with IL-2 prior to transfer prevents the decrease of T reg and improves their in vitro suppressive ability resulting in reduced TEC H/P in recipient mice. Therefore, culturing splenocytes improves transfer of TEC H/P by reducing the number of T reg and IL-2 inhibits transfer by preserving T reg number and function.

Reduced effectiveness of CD4+Foxp3+ regulatory T cells in CD28-deficient NOD.H-2h4 mice leads to increased severity of spontaneous autoimmune thyroiditis.

NOD.H-2h4 mice given NaI in their drinking water develop iodine-accelerated spontaneous autoimmune thyroiditis (ISAT) with chronic inflammation of the thyroid by T and B cells and production of anti-mouse thyroglobulin (MTg) autoantibody. CD28(-/-) NOD.H-2h4 mice, which have reduced numbers of CD4(+)Foxp3(+) regulatory T cells (Tregs), were developed to examine the role of Tregs in ISAT development. CD28(-/-) NOD.H2-h4 mice develop more severe ISAT than do wild-type (WT) mice, with collagen deposition (fibrosis) and low serum T4. CD28(-/-) mice have increased expression of proinflammatory cytokines IFN-γ and IL-6, consistent with increased mononuclear cell infiltration and tissue destruction in thyroids. Importantly, transferring purified CD4(+)Foxp3(+) Tregs from WT mice reduces ISAT severity in CD28(-/-) mice without increasing the total number of Tregs, suggesting that endogenous Tregs in CD28(-/-) mice are functionally ineffective. Endogenous CD28(-/-) Tregs have reduced surface expression of CD27, TNFR2 p75, and glucocorticoid-induced TNFR-related protein compared with transferred CD28(+/+) Tregs. Although anti-MTg autoantibody levels generally correlate with ISAT severity scores in WT mice, CD28(-/-) mice have lower anti-MTg autoantibody responses than do WT mice. The percentages of follicular B cells are decreased and those of marginal zone B cells are increased in spleens of CD28(-/-) mice, and they have fewer thyroid-infiltrating B cells than do WT mice. This suggests that CD28 deficiency has direct and indirect effects on the B cell compartment. B cell-deficient (B(-/-)) NOD.H-2h4 mice are resistant to ISAT, but CD28(-/-)B(-/-) mice develop ISAT comparable to WT mice and have reduced numbers of Tregs compared with WT B(-/-) mice.

Agonistic anti-CD40 induces thyrocyte proliferation and promotes thyroid autoimmunity by increasing CD40 expression on thyroid epithelial cells.

CD40 is expressed on cells of the immune system and in some tissues that are targets for autoimmune-mediated damage. It is not known if CD40 expression in target tissues plays a role in the pathology of autoimmune diseases. This study shows that agonistic anti-CD40 induces strong and sustained proliferation of thyroid epithelial cells (TECs), or thyrocytes, in IFN-γ(-/-) autoimmune-prone NOD and NOD.H-2h4 mice. TEC proliferation is accompanied by greatly increased expression of CD40 on TECs, development of fibrosis and hypothyroidism, and increased expression of proinflammatory molecules in thyroids. Bone marrow chimera experiments indicate that TEC expression of CD40 is required for anti-CD40-induced TEC proliferation, but lymphoid cells do not have to express CD40. TEC proliferation is reduced in wild-type mice given anti-CD40, presumably because they produce IFN-γ, which inhibits TEC proliferation. CD40 also increases on TECs during development of an autoimmune thyroid disease characterized by TEC hyperproliferation that develops spontaneously in IFN-γ(-/-) NOD.H-2h4 mice. TEC hyperproliferation development is accelerated in mice given agonistic anti-CD40. These studies provide new information regarding the role of target tissue expression of CD40 in development of autoimmunity and suggest that use of agonistic anti-CD40 for tumor therapy could result in autoimmune disease.

Transient depletion of CD4+ CD25+ regulatory T cells results in multiple autoimmune diseases in wild-type and B-cell-deficient NOD mice.

Approximately 80% of female wild-type non-obese diabetic (WT NOD) mice spontaneously develop diabetes, whereas B-cell-deficient (B(-/-)) NOD mice are resistant to diabetes. B(-/-) mice are also resistant to other spontaneous and experimentally induced autoimmune diseases, including arthritis, systemic lupus erythematosus, Sjögren syndrome and thyroiditis. Under normal conditions, activation of self-reactive T cells in the periphery is limited by CD4(+) CD25(+) natural regulatory T (Treg) cells. B(-/-) NOD.H-2h4 mice, normally resistant to spontaneous autoimmune thyroiditis (SAT), develop SAT when Treg cells are depleted, suggesting that Treg cells are preferentially activated when autoantigen is initially presented by non-B-cell antigen-presenting cells. To test the hypothesis that increased Treg cell activity in B(-/-) mice contributes to their resistance to other autoimmune diseases, WT and B(-/-) NOD mice were given anti-CD25 to transiently deplete CD4(+) CD25(+) Treg cells. The WT and B(-/-) NOD mice given anti-CD25 developed diabetes much earlier than WT mice given rat IgG, whereas rat IgG-treated B(-/-) mice did not develop diabetes. Treg-cell-depleted mice had increased lymphocyte infiltration of the pancreas, salivary glands and thyroid compared with controls given rat IgG. These results are consistent with the hypothesis that resistance of B-cell-deficient NOD mice to several autoimmune diseases is due to the activity of Treg cells.

Agonistic anti-CD40 promotes early development and increases the incidence of severe thyroid epithelial cell hyperplasia (TEC H/P) in CD4-/- mice.

IFN-γ-/-NOD.H-2h4 mice develop thyroid epithelial cell hyperplasia (TEC H/P) characterised by abnormal proliferation of thyrocytes and infiltration of thyroids by CD4+ and CD8+ T cells, macrophages and dendritic cells. CD8+ T cells from mice with severe TEC H/P transfer similar lesions to SCID recipients, whereas CD4+ T cells transfer mild TEC H/P. CD4- and CD8- deficient IFN-γ-/-NOD.H-2h4 mice were generated to determine if CD4+ T cells were required for initial activation of the CD8+ T cells that transfer TEC H/P. After 6-8 months on NaI water, only 2 of 60 CD8-/- mice developed severe TEC H/P, whereas 31 of 101 CD4-/- mice developed severe TEC H/P and fibrosis comparable in severity to that of IFN-γ-/- mice. However, splenocytes from CD4-/- mice with severe TEC H/P did not effectively transfer severe TEC H/P to SCID recipients. When CD4-/- donors were given agonistic anti-CD40 mAb, most developed severe TEC H/P and their cells transferred severe TEC H/P to SCID recipients. These results indicate that agonistic anti-CD40 can provide an important signal for activation of autoreactive CD8+ T cells that transfer severe TEC H/P. Therefore, targeting or blocking CD40 could provide effective therapy for diseases involving hyperplasia and fibrosis mediated by CD8+ T cells.

Transient depletion of B cells in young mice results in activation of regulatory T cells that inhibit development of autoimmune disease in adults.

B-cell depletion therapy can be effective for treating B-cell lymphomas as well as many human and murine autoimmune diseases. B-cell-deficient mice are normally resistant to spontaneous autoimmune thyroiditis (SAT), but they develop SAT if regulatory T cells are transiently depleted during the first 3-6 weeks after birth. This was also a critical time when B-cell depletion effectively inhibited development of SAT in adult mice. The current study was undertaken to test the hypothesis that transient depletion of B cells using anti-CD20 would be sufficient to suppress SAT if B cells were depleted early in life and that inhibition of SAT would be due to the activity of Treg that functioned most effectively when B cells were absent or low. The results presented here support this hypothesis and indicate that development of autoimmune disease in adults is effectively inhibited when anti-CD20 is administered 1-3 weeks after birth. After 3 weeks, transient B-cell depletion is no longer effective, and B-cell depletion must be maintained to effectively suppress autoimmune disease. B-cell depletion in 1- to 3-week-old mice depletes all B-cell subsets, whereas B-cell depletion initiated in adults spares many marginal zone B cells. Following early B-cell depletion, splenic Treg increase in number, and depletion of Treg reverses the inhibitory effect of anti-CD20 on disease development. Early transient depletion of B cells could be useful for preventing autoimmune disease in individuals at high risk for developing autoimmune diseases as adults.

TGF-ß promotes proliferation of thyroid epithelial cells in IFN-γ(-/-) mice by down-regulation of p21 and p27 via AKT pathway.

IFN-γ(-/-) NOD.H-2h4 mice develop an autoimmune disease characterized by hyperplasia and proliferation of thyroid epithelial cells (TEC H/P). Proliferating TECs produce TGF-ß, and IFN-γ inhibits TEC H/P. In the present study, cultured TECs were used to directly determine the mechanisms by which these cytokines act on TECs to result in proliferation or inhibition of proliferation. With TECs from IFN-γ(-/-) NOD.H-2h4 mice or mice expressing the dominant negative TGF-ß type II receptor on TECs, TGF-ß was shown to promote TEC proliferation and IFN-γ was shown to inhibit TEC proliferation in vitro. TGF-ß may promote TEC proliferation by down-regulating antiproliferative molecules p21 and p27, whereas IFN-γ may inhibit proliferation by up-regulating antiproliferative molecules p18 and p21 and down-regulating the pro-proliferative molecule cyclin D. Inhibition of AKT abolished the effect of TGF-ß on p21 and p27, resulting in similar proliferation of TGF-ß-treated and control TECs. Increased expression of proliferating cell nuclear antigen (PCNA), TGF-ß, and p-AKT and decreased expression of p21 and p27 by proliferating TECs correlated with the proliferative state of TEC H/P. Taken together, the results suggest that TGF-ß promotes TEC proliferation by down-regulating p21 and p27 via the AKT pathway in IFN-γ(-/-) NOD.H-2h4 mice, which may have significant implications for development of effective therapeutic strategies targeting the TGF-ß and AKT pathways for treatment of hyperplasia and/or neoplasia.

Effect of transgenic overexpression of FLIP on lymphocytes on development and resolution of experimental autoimmune thyroiditis.

In our previous studies, resolution of granulomatous experimental autoimmune thyroiditis (G-EAT) was promoted when thyroid epithelial cells were protected from Fas-mediated apoptosis due to transgenic overexpression of FLIP. We hypothesized that if FLIP were overexpressed on lymphocytes, CD4(+) effector cells would be protected from Fas-mediated apoptosis, and resolution would be delayed. To test this hypothesis, we generated transgenic (Tg) mice overexpressing FLIP under the CD2 promoter. Transgenic FLIP was expressed on CD4(+) and CD8(+) T cells and B cells. Transgenic overexpression of FLIP protected cultured splenocytes from Fas-mediated, but not irradiation-induced, apoptosis in vitro. Unexpectedly, Tg(+) donor cells transferred minimal G-EAT, which was partially overcome by depleting donor CD8(+) T cells. When Tg(+) and Tg(-) donors transferred equivalent disease, G-EAT resolution was delayed in FLIP transgenic mice. However, CD2-FLIP Tg(+) donors often transferred less severe G-EAT, even after depletion of CD8(+) T cells. This influenced the rate of G-EAT resolution, resulting in little difference in G-EAT resolution between groups. Tg(+) mice always had reduced anti-mouse thyroglobulin autoantibody responses, compared with Tg(-) littermates, presumably because of FLIP overexpression on B cells. These results suggest that effects of transgenic FLIP on a particular autoimmune disease vary, depending on what cells express the transgene and whether those cells are effector cells or if they function to modulate disease.

CD8+ T cells induce thyroid epithelial cell hyperplasia and fibrosis.

CD8(+) T cells can be important effector cells in autoimmune inflammation, generally because they can damage target cells by cytotoxicity. This study shows that activated CD8(+) T cells induce thyroid epithelial cell hyperplasia and proliferation and fibrosis in IFN-γ(-/-) NOD.H-2h4 SCID mice in the absence of CD4(+) T cells. Because CD8(+) T cells induce proliferation rather than cytotoxicity of target cells, these results describe a novel function for CD8(+) T cells in autoimmune disease. In contrast to the ability of purified CD8(+) T cells to induce thyrocyte proliferation, CD4(+) T cells or CD8 T cell-depleted splenocytes induced only mild thyroid lesions in SCID recipients. T cells in both spleens and thyroids highly produce TNF-α. TNF-α promotes proliferation of thyrocytes in vitro, and anti-TNF-α inhibits development of thyroid epithelial cell hyperplasia and proliferation in SCID recipients of IFN-γ(-/-) splenocytes. This suggests that targeting CD8(+) T cells and/or TNF-α may be effective for treating epithelial cell hyperplasia and fibrosis.

Antibodies to thyroid peroxidase arise spontaneously with age in NOD.H-2h4 mice and appear after thyroglobulin antibodies.

Hashimoto's thyroiditis, a common autoimmune disease, is associated with autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO). TPO, unlike abundant and easily purified Tg, is rarely investigated as an autoantigen in animals. We asked whether antibodies (Abs) develop to both TPO and Tg in thyroiditis that is induced (C57BL/6 and DBA/1 mice) or arises spontaneously (NOD.H-2h4 mice). Screening for TPOAbs was performed by flow cytometry using mouse TPO-expressing eukaryotic cells. Sera were also tested for binding to purified mouse Tg and human TPO. The antibody data were compared with the extent of thyroiditis. Immunization with mouse TPO adenovirus broke self-tolerance to this protein in C57BL/6 mice, but thyroiditis was minimal and TgAbs were absent. In DBA/1 mice with extensive granulomatous thyroiditis induced by Tg immunization, TPOAbs were virtually absent despite high levels of TgAbs. In contrast, antibodies to mouse TPO, with minimal cross-reactivity with human TPO, arose spontaneously in older (7-12 months) NOD.H-2h4 mice. Unexpectedly, TgAbs preceded TPOAbs, a time course paralleled in relatives of probands with juvenile Hashimoto's thyroiditis. These findings demonstrate a novel aspect of murine and human thyroid autoimmunity, namely breaking B cell self-tolerance occurs first for Tg and subsequently for TPO.

Transgenic expression of TGF-beta on thyrocytes inhibits development of spontaneous autoimmune thyroiditis and increases regulatory T cells in thyroids of NOD.H-2h4 mice.

Transgenic NOD.H-2h4 mice expressing TGF-beta under control of the thyroglobulin promoter were generated to assess the role of TGF-beta in the development of thyrocyte hyperplasia. In contrast to nontransgenic littermates, which develop lymphocytic spontaneous autoimmune thyroiditis (L-SAT), all TGF-beta transgenic (Tg) mice given NaI water for 2-7 mo developed thyroid lesions characterized by severe thyroid epithelial cell hyperplasia and proliferation, with fibrosis and less lymphocyte infiltration than in nontransgenic mice. Most Tg mice produced less anti-mouse thyroglobulin autoantibody than did wild type (WT) mice. T cells from Tg and WT mice were equivalent in their ability to induce L-SAT after transfer to SCID or TCRalpha(-/-) mice. WT lymphocytes could transfer experimental autoimmune thyroiditis or L-SAT to Tg mice, indicating that the transgenic environment did not prevent migration of lymphocytes to the thyroid. Thyroids of Tg mice had higher frequencies of Foxp3(+) regulatory T cells (Tregs) compared with nontransgenic WT mice. Transient depletion of Tregs by anti-CD25 resulted in increased infiltration of inflammatory cells into thyroids of transgenic mice. Treg depletion also resulted in increased anti-mouse thyroglobulin autoantibody responses and increased expression of IFN-gamma and IFN-gamma-inducible chemokines in thyroids of Tg mice. The results suggest that spontaneous autoimmune thyroiditis is inhibited in mice expressing transgenic TGF-beta on thyrocytes, at least in part, because there is an increased frequency of Tregs in their thyroids.