Margret Ehlers

Evidence of a Combined Cytotoxic Thyroglobulin and Thyroperoxidase Epitope-Specific Cellular Immunity in Hashimoto's Thyroiditis

CONTEXT Hashimotos thyroiditis (HT) is a common autoimmune disease leading to thyroid destruction due to lymphocytic infiltration. Only rare data are available regarding the recognition of specific cellular antigens, e.g. of thyroperoxidase (TPO) and thyroglobulin (Tg). OBJECTIVE The aim of this study was to quantify and characterize TPO- and Tg-epitope-specific CD8-positive T cells of HT patients. DESIGN Six different human leukocyte antigen (HLA)-A2 restricted, TPO- or Tg-specific tetramers were synthesized and used for measuring CD8-positive T cells in HT patients and controls. RESULTS The frequency of peripheral TPO- and Tg-specific CD8-positive T cells was significantly higher in HLA-A2-positive HT patients (2.8 ± 9.5%) compared with HLA-A2-negative HT patients (0.5 ± 0.7%), HLA-A2-positive nonautoimmune goiter patients (0.2 ± 0.4%), and HLA-A2-positive healthy controls (0.1 ± 0.2%). The frequency of Tg-specific T cells (3.0%) was very similar to those of TPO-specific CD8-positive T cells (2.9%). Subgroup analyses revealed a steady increase of the number of epitope-specific CD8-positive T cells from 0.6 ± 1.0% at initial diagnosis up to 9.4 ± 18.3% in patients with long-lasting disease. Analyses of the number of thyroid-infiltrating cells as well as the cytotoxic capacity revealed a similar picture for TPO- and Tg-specific T cells. CONCLUSION We here report for the first time that both antigens, TPO and Tg, are recognized by CD8-positive T cells and are involved in the thyroid destruction process leading to clinical disease manifestation.

Hashimoto's thyroiditis and papillary thyroid cancer: are they immunologically linked?

Hashimotos thyroiditis (HT) is the most common autoimmune disease in humans frequently leading to hypothyroidism. HT is characterized by a cellular immune response with lymphatic infiltration of the thyroid gland by T and B cells, as well as by a humoral immune response leading to specific antibody production. The synchronous appearance of HT and papillary thyroid cancer (PTC) indicates an immunological link between the two entities. Three different pathomechanisms may be postulated, including preexisting autoimmunity leading to malignancy due to inflammation, immunity towards preexisiting tumor cells leading to specific autoimmunity, and immune tolerance leading to malignancy despite (auto)immunity. In this article we review data describing these potential mechanisms that might lead to the synchronous appearance of HT and PTC.

MicroRNAs miR-146a1, miR-155_2, and miR-200a1 Are Regulated in Autoimmune Thyroid Diseases

Graves’ disease (GD) and Hashimoto’s thyroiditis (HT) are the most common autoimmune thyroid diseases (AITDs). The exact etiology of the immune response in both is still unknown. MicroRNAs (miRNAs) constitute a recently discovered family of small RNAs that control gene expression. Accumulating data suggest that miRNAs crucially control innate and adaptive immune responses, and implicate some miRNAs as having an important role in the pathophysiology of immunity and autoimmunity (1). To the best of our knowledge, there are currently no data on the involvement of miRNA in AITDs. We have determined the levels of some key immunoregulatory miRNAs in the thyroid glands of AITD patients and healthy controls. We investigated the thyroid gland fine-needle aspiration biopsies of patients with HT and GD. There were 28 unrelated Caucasians who were included in this study. The control groups comprised 9 Caucasians without AITDs; there were 9 patients with GD in the GD, and there were 10 patients with HT in the HT group. Ultrasound-guided fine-needle aspiration biopsies were performed using a 20-gauge needle. Total RNA, including miRNA, was extracted from thyroid tissue samples and reverse transcribed with an miRNeasy Mini Kit (Qiagen GmbH). Semiquantitative TaqMan PCR was performed in an ABI PRISM 7700 Sequence Detector (PE Applied Biosystems) using the miScript SYBR Green PCR Kit (Qiagen GmbH). The specific miScript Primer Assays (Qiagen GmbH) were used for the amplification of 13 miRNAs: miR-34a, miR-143_1, miR-143_1*, miR-146a_1, miR-155*_1, miR-155_2, miR-181a*_1, miR-181a2*, miR-181b1, miR-200a1, miR-200a*2, miR-200a1, and miR-1238. All data were analyzed using the nonparametric Kruskal–Wallis test. Results with p < 0.05 were considered statistically significant. Bonferroni-corrected Mann– Whitney U-tests were used as post hoc tests (see Supplementary Data, available online at www.liebertpub.com/thy). In the thyroid tissue of the GD group, miR-146a1 was significantly decreased in comparison to the control group (mean relative expression 5.17 vs. 8.37, respectively, p = 0.019). In the HT group, miR-155_2 was significantly decreased in comparison to the control group (8.30 vs. 11.20, respectively, p = 0.001), and miR-200a1 was significantly increased (12.02 vs. 8.01, p = 0.016). There were no significant differences between the HT and GD groups (see Supplementary Data) or for the other miRNAs tested. Our pilot study was undertaken to evaluate the influence of candidate miRNAs in AITDs that have been suspected of being involved in immunoregulation based on previously published observations (1–4). We found a significant decrease of miR-146a1 in the thyroid tissue of a patient with GD. miR-146a has been reported to be differentially regulated in other autoimmune diseases. Nakasa et al. demonstrated that miR-146a was highly expressed in rheumatoid arthritis (RA) synovial tissue compared to normal synovial tissue (2). This is in contrast to the decrease of miR-146a in our study, and is consistent with the perception that RA is characterized by a predominance of Th1-cytokines, while GD is characterized by the production of Th2-cytokines in thyroid tissue-emigrating mononuclear cells. Furthermore, we report here that miR200a1 is increased in the thyroid tissue of HT patients, but not in that of patients with GD. This further substantiates the findings by Glinsky, who found in an SNP-guided miRNA map that miR-200, miR-34a, miR-143, and miR-1238 were associated with AITDs in general and GD in particular (4). To date, the potential role of miR-200a1 is not understood and warrants further research. This is the first report that there is a significant decrease of miR-155_2 in the thyroid tissue of patients with HT. This miRNA was previously shown to possess important functions in the mammalian immune system by Thai et al. (3). The other miRNA candidates tested did not significantly differ between AITDs, even though evidence exists for their involvement in immunoregulation. We assume that the reported miRNA variations in thyroid tissue are caused by infiltrating activated lymphocytes, because—according to additionally performed microscopy of fine-needle aspirations—these cells are dominant.

Localization and regulation of pancreatic selenoprotein P

Progressive loss of pancreatic β-cell mass is a crucial feature of type 2 diabetes mellitus. As β-cells express very low amounts of the antioxidant enzymes catalase and glutathione peroxidase (GPx), they appear to be particularly vulnerable to oxidative damage in the pathogenesis of diabetes. Here, we investigated the pancreatic expression pattern and regulation of selenoprotein P (Sepp1), which may serve as an additional antioxidant enzyme inside and outside of cells. Sepp1 was detected in rodent pancreas by immunofluorescence and real-time RT-PCR. Regulation of Sepp1 biosynthesis in INS-1 rat insulinoma cells was investigated by real-time RT-PCR, luciferase gene reporter assay, and immunoblotting. Sepp1 and Gpx1 gene expressions in rat pancreas were 58 and 22% respectively of the liver values. Pancreatic Sepp1 expression was restricted to the endocrine tissue, with Sepp1 being present in the α- and β-cells of mouse islets. In INS-1 insulinoma cells, Sepp1 expression was stimulated by the selenium compound sodium selenate and diminished in the presence of high glucose (16.7 vs 5  mM) concentrations. Sepp1 mRNA stability was also lowered at 16.7  mM glucose. Moreover, Sepp1 mRNA levels were decreased in isolated murine islets cultured in high-glucose (22  mM) medium compared with normal glucose (5.5  mM) medium. Pancreatic Sepp1 expression was elevated upon treatment of mice with the β-cell toxin streptozotocin. This study shows that pancreatic islets express relatively high levels of Sepp1 that may fulfill a function in antioxidant protection of β-cells. Downregulation of Sepp1 expression by high glucose might thus contribute to glucotoxicity in β-cells.

Immunoregulatory Natural Killer Cells Suppress Autoimmunity by Down-Regulating Antigen-Specific CD8+ T Cells in Mice

Natural killer (NK) cells belong to the innate immune system. Besides their role in antitumor immunity, NK cells also regulate the activity of other cells of the immune system, including dendritic cells, macrophages, and T cells, and may, therefore, be involved in autoimmune processes. The aim of the present study was to clarify the role of NK cells within this context. Using two mouse models for type 1 diabetes mellitus, a new subset of NK cells with regulatory function was identified. These cells were generated from conventional NK cells by incubation with IL-18 and are characterized by the expression of the surface markers CD117 (also known as c-Kit, stem cell factor receptor) and programmed death (PD)-ligand 1. In vitro analyses demonstrated a direct lysis activity of IL-18-stimulated NK cells against activated insulin-specific CD8(+) T cells in a PD-1/PD-ligand 1-dependent manner. Flow cytometry analyses revealed a large increase of splenic and lymphatic NK1.1(+)/c-Kit(+) NK cells in nonobese diabetic mice at 8 wk of age, the time point of acceleration of adaptive cytotoxic immunity. Adoptive transfer of unstimulated and IL-18-stimulated NK cells into streptozotocin-treated mice led to a delayed diabetes development and partial disease prevention in the group treated with IL-18-stimulated NK cells. Consistent with these data, mild diabetes was associated with increased numbers of NK1.1(+)/c-Kit(+) NK cells within the islets. Our results demonstrate a direct link between innate and adaptive immunity in autoimmunity with newly identified immunoregulatory NK cells displaying a potential role as immunosuppressors.

Advances in cellular therapy for the treatment of thyroid cancer.

Up to now, there are no curative therapies available for the subset of metastasized undifferentiated/anaplastic thyroid carcinomas. This review describes the possible use of immunocompetent cells which may help to restore the antitumor immune recognition for treating an existing tumor or preventing its recurrence. The most prominent experimental strategy is the use of dendritic cells (DCs) which are highly potent in presenting tumor antigens. Activated DCs subsequently migrate to draining lymph nodes where they present antigens to naïve lymphocytes and induce cytotoxic T cells (CTL). Alternatively to DC therapy, adoptive cell transfer may be performed by either using natural killer cells or ex vivo maturated CTLs. Within this review article we will focus on recent advances in the understanding of anti-tumor immune responses, for example, in thyroid carcinomas including the advances which have been made for the identification of potential tumor antigens in thyroid malignancies.

MicroRNA expressions in PMBCs, CD4+, and CD8+ T-cells from patients suffering from autoimmune Addison's disease.

Autoimmune Addisons disease (AD) is a rare but potentially life threatening disease. The exact etiology of the immune response to the adrenal gland is still unknown. MicroRNAs (miRNAs) critically control gene-expression and play an important role in regulating the immune response. The aim of this study was to determine key immunoregulatory miRNAs influencing autoimmune adrenal insufficiency. For this purpose selected miRNAs were amplified by a semiquantitative SYBR Green PCR from blood mononuclear cells and after purification from CD4+ and CD 8+ cells of 6 patients with autoimmune adrenal insufficiency and 10 healthy controls. In CD4+ T-cells miRNA 181a*_1 (18.02 in AD vs. 11.99 in CG, p=0.0047) is significantly increased whereas miRNA 200a_1 (12.48 in AD vs. 19.40 in CG, p=0.0003) and miRNA 200a_2* (8.59 in AD vs. 17.94 in CG, p=0.0160) are significantly decreased. miRNA 200a_1 (12.37 in AD group vs. 18.12 in control group, p=0.001) and miRNA 200a_2* (10.72 in AD group vs. 17.84 in control group, p=0.022) are also significantly decreased in CD8+ T-cells. This study could show for the first time a significant change of three defined miRNAs in PBMCs, CD4+, and CD8+ T-cells of autoimmune AD patients in vivo. These data may help to better understand the cause of the autoimmune processes leading to autoimmune AD. They extend our very limited knowledge concerning miRNAs in autoimmune Addisons disease.

Increased numbers of tumor-lysing monocytes in cancer patients.

Lymphatic infiltration is a well known phenomenon in different tumors including endocrine malignancies. However, little is known about the role of antigen-presenting cells and T cell activation in this context. The aim of our study was to investigate the quantity and function of CD14+/CD56+ monocytes in tumor patients including endocrine malignancies. First, these cells were characterized in peripheral blood of endocrine and non-endocrine cancer patients as well as in tumor tissue samples. Cancer patients had in mean 3.7 times more CD14+/CD56+ monocytes in the peripheral blood compared to healthy controls (p≤0.0001), while the highest frequencies were seen in patients with heavy tumor load. Importantly, these cells additionally expressed several NK cell markers. A proof of CD14+/CD56+ infiltrations into papillary thyroid carcinoma was shown by immunohistochemical analyses. Functional analyses revealed an apoptosis inducing capacity in vitro after IFN-α re-stimulation. Our data indicate the importance of tumor-lysing monocytes in antitumor immunity.

Endocrine organs under the control of the immune system: potential implications for cellular therapies.

Within the last couple of years much knowledge has been gained in understanding the immune interactions in endocrine diseases including endocrine malignancies and autoimmune diseases. The major players within the innate immune system represent NK cells. This review describes that these cells directly lyse tumor cells and promote the activity of other cells of the immune system, including dendritic cells (DCs), macrophages, Th1 cells, and cytotoxic T-lymphocytes (CTLs). NK cells may also be involved in the initiation of autoimmunity as they may accumulate in target organs of certain autoimmune diseases. On the other hand, there are cells of the adaptive immune system including antigen-presenting DCs and T cells with helper and effector function, which are responsible for a directed immune response. Within this review, we present an overview on the role of all these cell populations in endocrine disease and the potential use of such cells for immunotherapy in different endocrine diseases and refer to experimental settings as well as clinical studies.

Chromogranin A as potential target for immunotherapy of malignant pheochromocytoma.

Currently, no effective treatment for malignant pheochromocytoma exists. The aim of our study was to investigate the role of chromogranin A (CgA) as a specific target molecule for immunotherapy in a murine model for pheochromocytoma. Six amino acid-modified and non-modified CgA peptides were used for dendritic cell vaccination. Altogether, 50 mice received two different CgA vaccination protocols; another 20 animals served as controls. In vitro tetramer analyses revealed large increases of CgA-specific cytotoxic T cells (CTL) in CgA-treated mice. Tumors of exogenous applied pheochromocytoma cells showed an extensive infiltration by CD8+ T cells. In vitro, CTL of CgA-treated mice exhibited strong MHC I restricted lysis capacities towards pheochromocytoma cells. Importantly, these mice showed strongly diminished outgrowth of liver tumors of applied pheochromocytoma cells. Our data clearly demonstrate that CgA peptide-based immunotherapy induces a cytotoxic immune response in experimental pheochromocytoma, indicating potential for therapeutic applications in patients with malignant pheochromocytoma.