Thyroid Lymphocytic Infiltration Research Articles

A Flexible Mouse Model of Autoimmune Thyroiditis Induced by Immunization with an Adenovirus Containing Full-Length Thyroglobulin cDNA.

The main challenge in the "post-GWAS" era is to determine the functional meaning of genetic variants and their contribution to disease pathogenesis. Development of suitable mouse models is critical because disease susceptibility is triggered by complex interactions between genetic, epigenetic, and environmental factors that cannot be modeled by in vitro models. Thyroglobulin (TG) is a key gene for autoimmune thyroid disease (AITD) and several single nucleotide polymorphisms (SNPs) in the TG coding region have been associated with AITD. The classical model of experimental autoimmune thyroiditis (EAT), based on immunization of genetically susceptible mouse strains with purified TG protein in adjuvant, does not allow testing the impact of TG sequence variants on the development of autoimmune thyroiditis. Here we describe a protocol for the induction of EAT by immunization of mice susceptible to thyroiditis with an adenovirus vector carrying full-length human TG cDNA (Ad-TG EAT). We also provide support protocols for evaluation of autoimmune thyroiditis including serological assessment of TG antibodies, in vitro splenocyte proliferation assay and cytokines secretion, thyroid histology, and evaluation of thyroid lymphocytic infiltration by immunostaining. This protocol for EAT induction allows manipulation of the TG cDNA to introduce variants associated with AITD, enabling the testing of the functional effects of susceptible variants and their haplotypes on the immunogenicity of TG. Furthermore, the Ad-TG EAT mouse model is a valuable model for studying the interactions of the TG variants with non-genetic factors influencing AITD development (e.g., cytokines, iodine exposure) or with variants of other susceptible genes (e.g., HLA-DRβ1). © 2024 Wiley Periodicals LLC. Basic Protocol: Development of a mouse model of autoimmune thyroiditis induced by immunization with adenovirus containing full-length thyroglobulin cDNA Support Protocol 1: Splenocytes isolation Support Protocol 2: T cell stimulation and carboxyfluorescein diacetate succinimidyl ester (CFSE) based cell proliferation assay Support Protocol 3: Cytokine assays: measuring levels of interferon gamma (IFNγ) and interleukins IL-2, IL-4, and IL-10 in splenocyte supernatants Support Protocol 4: Evaluating thyroid histology and infiltration with immune cells: hematoxylin-eosin staining of mice thyroid glands Support Protocol 5: Immunohistochemistry of thyroid tissues: Immunofluorescence protocol of paraffin-embedded thyroid sections Support Protocol 6: Anti-thyroglobulin antibody measurement in mice sera by enzyme-linked immunosorbent assay (ELISA).

Thyroid hormones and minerals in immunocorrection of disorders in autoimmune thyroid diseases.

Thyroid hormones and essential elements iodine (I), selenium (Se), iron (Fe), copper (Cu), zinc (Zn), calcium (Ca), magnesium (Mg), etc. play an important role in the work of many organs and systems of the body, including the immune system and the thyroid gland, and a violation of their supply can be the cause of pathological changes in them. In pathology, the interaction between thyroid hormones (TG), minerals and the immune system is disturbed. The review of the literature examines the immunomodulatory role of TG, minerals, their properties, and their participation in the pathogenesis of autoimmune thyroid diseases (AITD). The study of the relationship between the excess or deficiency of minerals and AITD is described. The basis of the development of AITD - Hashimoto's thyroiditis (HT), Graves' disease (GD), Graves' ophthalmopathy (GO) is the loss of immune tolerance to thyroid antigens - thyroid peroxidase (TPO), thyroglobulin (Tg) and thyroid-stimulating hormone receptor (TSH-R). Immune-mediated mechanisms - production of autoantibodies to thyroid antigens and lymphocytic thyroid infiltration - are involved in the pathogenesis of AITD. Insufficiency of regulatory T cells (Treg) and regulatory B cells (Breg), imbalance between Th17-lymphocytes and Treg-lymphocytes, abnormal production of pro-inflammatory cytokines has a significant influence on the progression of AITD. With AITD, the balance between oxidants and antioxidants is disturbed and oxidative stress (OS) occurs. The lack of modern effective pharmacological therapy of AITD prompted us to consider the mechanisms of influence, possibilities of immunocorrection of pathogenetic factors using TG, micro/macronutrients. In order to develop a more effective treatment strategy, as well as approaches to prevention, a critical analysis of the ways of immunotherapeutic use of dietary supplements of I, Se, Zn, Mg and other minerals in AITD was carried out.

OR19-5 Dissecting Thyroidal Pathways Activated by PD-L1 Blockade Identifies New Mechanisms for Thyroiditis Triggered by Immune Checkpoint Inhibitors

Abstract Programmed death-ligand 1 (PD-L1) is a strong immunosuppressor that inhibits T-cell activation by binding to its receptor (PD-1) on T-cells. Blocking the PD-L1/PD-1 interaction by monoclonal antibodies (mAbs) is an effective therapy for several cancers via activation of T-cells. Destructive thyroiditis and hypothyroidism often develop during anti-PD-L1 (αPD-L1) therapy but the pathogenic mechanisms are not known. We evaluated in vivo, the role of PD-L1 and its interactions with inflammatory triggers in the development of autoimmune thyroiditis (AIT). NOD.H-2h4 mice were treated with either αPD-L1 or isotype control (CTR) for two weeks and AIT evaluated after six weeks. αPD-L1 treated mice showed significantly higher Abs (p = 6×10e-4) and T-cell responses (p = 0.02) to thyroglobulin (Tg) compared with CTR mice. Serum fT4 levels were lower (p = 0.01) and TSH higher (p = 0.05) in αPD-L1 treated vs. CTR mice. Seven of the eight αPD-L1 treated mice (and none of the CTR mice) developed thyroid lymphocytic infiltration characterized by the presence of the CD4+, CD8+, FoxP3+ T- cells and B220+ B- cells. To gain insight into the pathways regulated by PD-L1 in the thyroid we assessed gene expression in the thyroid of αPD-L1 treated mice using RNA-seq. PD-L1 blockade was associated with upregulation of pathways of lymphocyte activation (p = 1.3×10e-5), lymphocyte proliferation (p = 2×10e-5), chemotaxis (p = 3.8×10e-12), and inflammatory response (p = 7.9×10e-7). Specifically, blocking PD-L1 was associated with upregulation of cytokine/chemokine genes that have a critical role in Th1 differentiation: IFNγ, IL-12, IL-15, CCL1 to CCL5, CD300, CXCL10, CXCL11. Ingenuity Pathway Analysis identified IFNγ and TNF as the top regulators of these activated gene networks. These results indicate that blockade of PD-L1 in thyroid promoted the Th1 mediator, IFNγ, inducing activation and proliferation of thyroidal CD4+ and CD8+ T cells, triggering thyroid tissue damage and AIT. Since PD-L1 mediates immune responses to inflammatory stressors (e.g. viral infections), we evaluated PD-L1 interaction with a known inflammatory trigger of AIT, interferon alpha (IFNα). Treatment with αPD-L1 + IFNα accelerated AIT in NOD.H-2h4 mice that developed high Tg Ab and T-cell responses and thyroidal lymphocytic infiltration only two weeks after treatment. Moreover, thyroiditis was more severe in mice receiving αPD-L1 + IFNα than αPD-L1 alone, manifesting by higher T-cell responses (p = 5×10e-3), and significantly more profound hypothyroidism. These results suggest that PDL-1 has a protective role in the thyroid against inflammatory stressors, and that its blockade exposes the thyroid to inflammatory damage. Therefore, in patients undergoing αPD-L1 therapy, inflammatory conditions (e.g. infections) can be a "second hit" that accelerates the onset of thyroiditis and augments thyroidal inflammation. Presentation: Monday, June 13, 2022 12:00 a.m. - 12:15 a.m.

The protective effect of iodide intake adjustment and 1,25(OH)2D3 supplementation in rat offspring following excess iodide intake.

Background:In this study, we aimed to investigate the effect of iodide intake adjustment, 1,25(OH)2D3 supplementation, or both, on the thyroid gland of rat offspring.Methods:The offspring of female rats administered 100 times the normal dose of iodide (100 HI; 750 μg/d) during pregnancy and lactation were divided into four different treatment groups. They were either having their iodide intake adjusted from 100 HI to normal iodide intake (7.5 μg/day) or supplemented with 25-hydroxy vitamin D3 [1,25(OH)2D3; 5 μg·kg−1·day−1], or both, for 4 weeks. Thyroid sodium pertechnetate (Na99mTcO4) uptake percentages were measured using single-photon emission computed tomography, while serum levels of free triiodothyronine (FT3), free thyroxine (FT4), thyroglobulin antibody (TgAb), thyroid peroxidase antibody (TPOAb), and vitamin D3 (VD3) were monitored using enzyme-linked immunosorbent assay. The messenger ribonucleic acid expression of interleukin (IL)-17A, interferon gamma (IFN-γ), and IL-10 in the thyroid gland was measured using quantitative real-time polymerase chain reaction, while the protein expression of thyroid-hormone-receptor α1 (TRα1) and thyroid-hormone-receptor β1 (TRβ1) in the thyroid gland was detected using Western blotting. Haematoxylin and eosin (H & E) and immunofluorescence staining were also used to assess thyroid follicular structure and lymphocytic infiltration in the thyroid glands.Results:The immunofluorescence staining showed CD4+ co-localized with TRβ1 or the vitamin D receptor in thyroid gland cells of rats that were continuously treated with 100 HI. Following iodide adjustment, 1,25(OH)2D3 supplementation, or both, an increase in serum levels of FT3, free thyroxine, and VD3, protein expression of TRα1 and TRβ1 in the thyroid gland cells, and Na99mTcO4 thyroid uptake percentages was observed. The mRNA expression levels of IL-17A and IFN-γ, decreased, while the mRNA expression levels of IL-10 increased in the thyroid cells of each treatment group, except the group with continuous 100 HI intake.Conclusion:Iodide adjustment, 1,25(OH)2D3 supplementation, or both may increase the serum levels of FT3, FT4, and VD3, as well as the protein expression levels of TRα1 and TRβ1, in thyroid cells. In addition, iodide adjustment, 1,25(OH)2D3 supplementation, or both, may potentially reverse the imbalance in pro-inflammatory and anti-inflammatory cytokines (IL-17A, IFN-γ, and IL-10) caused by 100 HI, which may be beneficial in improving Na99mTcO4 thyroid uptake percentages.

Thyroid Hemiagenesis in a Thyroiditis Prone Mouse Strain

Background: Thyroid hemiagenesis, a rare congenital condition detected by ultrasound screening of the neck, is usually not manifested clinically in humans. This condition has been reported in mice with hypothyroidism associated with induced deficiency in paired box 8 and NK2 homeobox 1, sonic hedgehog, or T-box 1. Unexpectedly, we observed thyroid hemiagenesis in NOD.H2^h4 mice, an unusual strain that spontaneously develops iodide enhanced thyroid autoimmunity but remains euthyroid. Objectives and Methods: First, to compare mice with thyroid hemiagenesis versus bilobed littermates for serum T4, autoantibodies to thyroglobulin (ELISA) and thyroid peroxidase (TPO; flow cytometry with eukaryotic cells expressing mouse TPO), gross anatomy, and thyroid histology; second, to estimate the percentage of mice with thyroid hemiagenesis in the NOD.H2^h4 mice we have studied over 6 years. Results: Thyroid hemiagenesis was observed in 3 of 1,025 NOD.H2^h4 mice (2 females, 1 male; 0.3%). Two instances of hemiagenesis were in wild-type females and one in a transgenic male expressing the human TSHR A-subunit in the thyroid. Two mice had very large unilobed glands, as in some human cases with this condition. Thyroid lymphocytic infiltration, serum T4, and the levels of thyroid autoantibodies were similar in mice with thyroid hemiagenesis and bilobed littermates. Conclusions: Unlike hypothyroidism associated with hemiagenesis in transcription factor knockout mice, hemiagenesis in euthyroid NOD.H2^h4 mice occurs spontaneously and is phenotypically similar to that occasionally observed in humans.

Prognostic significance of thyroglobulin antibody epitopes in differentiated thyroid cancer.

Thyroglobulin antibodies (TgAbs) are surrogate markers of disease recurrence or persistence in differentiated thyroid cancer (DTC). However, the prognostic significance of TgAb heterogeneity in DTC has not been investigated. To evaluate the relationship between TgAb epitope specificities and clinical outcomes in DTC patients. We studied 61 TgAb-positive patients with DTC, post-thyroidectomy and remnant ablation (7 males, 54 females; age-range 16-80 years, median follow-up duration 8.9 years). TgAb epitope reactivities were mapped using a panel of 10 thyroglobulin (Tg) monoclonal antibodies delineating six antigenic Tg clusters in competitive ELISA studies. Sera from 45 patients with Hashimoto's thyroiditis (HT) and 22 TgAb-positive healthy subjects served as autoimmune and healthy controls. Tg was measured by immunoradiometric assay (IRMA), electrochemiluminescence immunoassay (ECLIA), and RIA, while TgAbs was measured by ELISA and ECLIA methods. Samples from 26 DTC patients showed TgAb epitope restriction similar to HT patients, while 35 patients exhibited nonspecific reactivity comparable to healthy controls. DTC patients with epitope restriction had higher rates of recurrent/persistent disease (81% vs 17%, P < .001), higher median TgAb concentration (887.0 vs 82.0 kIU/L; P < .001), and a higher prevalence of thyroid lymphocytic infiltration (71.4% vs 26.8%; P < .001) compared to patients with nonspecific reactivity. Samples with epitope restriction also had a lower median Tg-IRMA/RIA ratio (3.0% vs 36.0%; P < .001) denoting greater degrees of Tg assay interference. TgAb epitope restriction is associated with a less favorable prognosis than nonspecific reactivity in DTC patients. TgAb epitope specificities may have prognostic value in DTC.

The prevalence of thyroid autoimmunity in patients with urticaria: a systematic review and meta-analysis.

Thyroid autoimmunity is the most common organ-specific autoimmune disorder, which is characterized by the production of thyroid autoantibodies and lymphocytic infiltration into the thyroid. The majority cases of chronic urticaria have unknown (idiopathic) causes, with about 30-40 % possibly having an autoimmune substrate. Considering that autoimmune factors may be the common features of both thyroid autoimmunity and urticaria, it is likely that both entities may coexist within the same patient. A number of studies have investigated the association between thyroid autoimmunity and urticaria. However, most of these studies are relatively small sample size, the power achieved in those studies was not sufficient to detect whether there is an association between urticaria and thyroid autoimmunity. The aim of this study is to combine primary data from all relevant studies to produce reliable estimates of the associations between thyroid autoantibodies and urticaria. Literature databases were searched including Medline, Embase, Web of Science, Chinese Wanfang, and CBM databases from January 1980 to December 2013. A total of 14,203 urticaria cases and 12,339 non-urticaria controls were included in this study. From these data, the odds ratio (OR) with 95% confidence interval (95% CI) was calculated. The meta-analysis results showed that the prevalence of positive thyroid autoantibodies in patients with urticaria was higher than non-urticaria controls (TgAb: OR 6.55, 95% CI 3.19-13.42, P<0.00001, I2=67%; TmAb: OR 4.51, 95% CI 2.78-7.33, P<0.00001, I2=47%; TPOAb: OR 8.71, 95% CI 6.89-11.01, P<0.00001, I2=20%, respectively). The results of this meta-analysis suggested that patients with urticaria were more likely to have thyroid autoimmunity than the control groups.

BRAF V600E assessment by pyrosequencing in fine needle aspirates of thyroid nodules with concurrent Hashimoto’s thyroiditis is a reliable assay

Detection of BRAF mutation in cytology specimens has been proposed as a diagnostic adjunctive tool in evaluation of thyroid nodules with indeterminate cytology findings. Concurrent papillary thyroid carcinoma and Hashimoto's thyroiditis (HT), a disease characterized by thyroid lymphocytic infiltration, is a frequent occurrence. A large lymphocytic infiltrate might reduce the sensitivity of methods employed to detect BRAF mutation in thyroid cytology specimens. To determine whether testing for BRAF mutational status in fine needle aspirates (FNA) is reliable also in the presence of HT lymphocytic infiltration, we assessed the BRAF status by direct sequencing and pyrosequencing in a series of FNAs with and without concomitant HT lymphocytic infiltration. We also performed the same assessment by pyrosequencing in the corresponding tissue samples. Pyrosequencing demonstrated to be more sensitive than direct sequencing. The percentage of mutant BRAF(V600E) alleles was higher in FNAs than in the corresponding tissues, probably because of the lower stromal contamination in FNA than in the sections. In the presence of lymphocytic infiltration, the percentage of mutant BRAF(V600E) alleles determined by pyrosequencing was higher in FNAs than in the corresponding tissue samples (P < 0.0001), indicating a minor lymphocytic contamination in FNA. The diagnostic value of BRAF(V600E) in inconclusive FNAs was not hampered by thyroid lymphocytic infiltration. These results indicate that BRAF(V600E) assessment by pyrosequencing is a reliable assay useful to refine inconclusive cytology of thyroid nodules also in the presence of concurrent HT.

Demonstration of Innate Immune Responses in the Thyroid Gland: Potential to Sense Danger and a Possible Trigger for Autoimmune Reactions

Autoimmune thyroid disease is an archetypal organ-specific autoimmune disorder that is characterized by the production of thyroid autoantibodies and lymphocytic infiltration into the thyroid. However, the underlying mechanisms by which specific thyroid antibodies are produced are largely unknown. Recent studies have shown that innate immune responses affect both the phenotype and the severity of autoimmune reactions. Moreover, it appears that even non-immune cells, including thyroid cells, have an ability to launch such responses. The aim of this study was to conduct a more detailed analysis of innate immune responses of the thyroid upon stimulation with various "non-self" and "self" factors that might contribute to the initiation of autoimmune reactions. We used rat thyroid FRTL-5 cells, human thyroid cells, and mice to investigate the effects of various pathogen-associated molecular patterns (PAMPs), danger-associated molecular patterns (DAMPs), and iodide on gene expression and function that were related to innate immune responses. RT-PCR analysis showed that both rat and human thyroid cells expressed mRNAs for Toll-like receptors (TLRs) that sensed PAMPs. Stimulation of thyrocytes with TLR ligands resulted in activation of the interferon-beta (IFN-β) promoter and the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)-dependent promoter. As a result, pro-inflammatory cytokines, chemokines, and type I interferons were produced. Similar activation was observed when thyroid cells were stimulated with double-stranded DNA, one of the typical DAMPs. In addition to these PAMPs and DAMPs, treatment of thyroid cells with high concentrations of iodide increased mRNA expression of various cytokines. We show that thyroid cells express functional sensors for exogenous and endogenous dangers, and that they are capable of launching innate immune responses without the assistance of immune cells. Such responses may relate to the development of thyroiditis, which in turn may trigger autoimmune reactions.

Breaking Tolerance in Transgenic Mice Expressing the Human TSH Receptor A-Subunit: Thyroiditis, Epitope Spreading and Adjuvant as a ‘Double Edged Sword’

Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a “double-edged sword”. On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the TSHR) displaying strong self-tolerance.

Progressive thyroid lymphocytic infiltration in a patient with chronic hepatitis

Progressive thyroid lymphocytic infiltration in a patient with chronic hepatitis

Enhanced Response to Mouse Thyroid-Stimulating Hormone (TSH) Receptor Immunization in TSH Receptor-Knockout Mice

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.

Papillary thyroid cancer, although strongly associated with lymphocitic infiltration on histology, is only weakly predicted by serum thyroid auto-antibodies in patients with nodular thyroid diseases

We evaluated the association between thyroid autoimmunity and thyroid cancer in a retrospective series of unselected thyroid nodules submitted to fine-needle aspiration (FNA) cytology. Anti-thyroid antibodies (TAb) were measured in patients with multinodular goiter (MNG) and single/isolated thyroid nodule (S/I) submitted to FNA. Thyroid lymphocytic infiltration (LI) on histology was studied in a subgroup of patients submitted to thyroidectomy; 13,021 patients were included: on cytology 622 had papillary thyroid cancer (c- PTC) and 12,399 benign thyroid nodular diseases (c-BTN). LI was evaluated in histological samples of 688 patients: 304 with PTC (h-PTC) and 384 with BTN (h-BTN). TAb prevalence was not different in c-BTN and c-PTC (38.7% vs 35.6%). TAb were more frequent in c-BTN than c-PTC in females with MNG (40.1% vs 32.5%, p=0.02), and in c-PTC than in c-BTN in males with S/I (31.2% vs 20.4%, p=0.02) and, although not significantly, in females younger than 30 yr (35.1% vs 30.7%). The frequency and severity of LI was significantly higher in h-PTC than h-BTN, both in MNG (82.5% vs 45.0%, p<0.001) and S/I (85.6% vs 71.0%, p<0.001), but a higher number of patients with h-PTC had negative circulating TAb, despite the presence of moderate/severe LI. TAb are weakly associated to PTC in males and young females, while they are more frequent in older females with BTN. The frequency and severity of LI is significantly higher in PTC than in BTN, but in cancer patients TAb are frequently negative, despite the evidence of histological thyroiditis. These data suggest that different kinds of immune response may be involved in PTC and BTN.

Role of the transgenic human thyrotropin receptor A-subunit in thyroiditis induced by A-subunit immunization and regulatory T cell depletion

Transgenic BALB/c mice that express intrathyroidal human thyroid stimulating hormone receptor (TSHR) A-subunit, unlike wild-type (WT) littermates, develop thyroid lymphocytic infiltration and spreading to other thyroid autoantigens after T regulatory cell (T(reg)) depletion and immunization with human thyrotropin receptor (hTSHR) adenovirus. To determine if this process involves intramolecular epitope spreading, we studied antibody and T cell recognition of TSHR ectodomain peptides (A-Z). In transgenic and WT mice, regardless of T(reg) depletion, TSHR antibodies bound predominantly to N-terminal peptide A and much less to a few downstream peptides. After T(reg) depletion, splenocytes from WT mice responded to peptides C, D and J (all in the A-subunit), but transgenic splenocytes recognized only peptide D. Because CD4(+) T cells are critical for thyroid lymphocytic infiltration, amino acid sequences of these peptides were examined for in silico binding to BALB/c major histocompatibility complex class II (IA-d). High affinity subsequences (inhibitory concentration of 50% < 50 nm) are present in peptides C and D (not J) of the hTSHR and mouse TSHR equivalents. These data probably explain why transgenic splenocytes do not recognize peptide J. Mouse TSHR mRNA levels are comparable in transgenic and WT thyroids, but only transgenics have human A-subunit mRNA. Transgenic mice can present mouse TSHR and human A-subunit-derived peptides. However, WT mice can present only mouse TSHR, and two to four amino acid species differences may preclude recognition by CD4+ T cells activated by hTSHR-adenovirus. Overall, thyroid lymphocytic infiltration in the transgenic mice is unrelated to epitopic spreading but involves human A-subunit peptides for recognition by T cells activated using the hTSHR.

DNA Hypermethylation Status of Multiple Genes in Papillary Thyroid Carcinomas

Objective: The aim of this study was hypermethylation of multiple genes for papillary thyroid carcinomas (PTCs). Methods: We examined 39 lesions using methylation-specific PCR to assess hypermethylation in genes, including p16^INK4a, p14^ARF, RB1, p27^Kip1and 0⁶-MGMT. Homozygous deletions of p16^INK4a and p14^ARF were investigated by differential PCR, all with reference to clinicopathological factors. Results: We foundmethylation of p16^INK4a in 35.9% (14/39); p14^ARF in 2.6% (1/39); RB1 in 23.1% (9/39); p27^Kip1 in 15.4% (6/39),and 0⁶-MGMT in 15.4% (6/39). Hypermethylation of at least one of these genes was apparent in 66.7% (26/39). Homozygous deletions of p14^ARF and p16^INK4a were detected in 7.7 (3/39) and 2.6% (1/39), respectively. In cases with p16^INK4a alterations, tumors were significantly increased. A history of chronic thyroid disease and lymphocytic infiltration was significantly associated with p14^ARF alterations, without regional lymph node metastases. Conclusions: Our data suggest that alterations in p16^INK4a, mainly hypermethylation, may be linked to tumor growth but not tumor development, while alterations in p14^ARF may contribute to the induction of chronic inflammation-related PTCs.

The Link between Graves’ Disease and Hashimoto’s Thyroiditis: A Role for Regulatory T Cells

Hyperthyroidism in Graves' disease is caused by thyroid-stimulating autoantibodies to the TSH receptor (TSHR), whereas hypothyroidism in Hashimoto's thyroiditis is associated with thyroid peroxidase and thyroglobulin autoantibodies. In some Graves' patients, thyroiditis becomes sufficiently extensive to cure the hyperthyroidism with resultant hypothyroidism. Factors determining the balance between these two diseases, the commonest organ-specific autoimmune diseases affecting humans, are unknown. Serendipitous findings in transgenic BALB/c mice, with the human TSHR A-subunit targeted to the thyroid, shed light on this relationship. Of three transgenic lines, two expressed high levels and one expressed low intrathyroidal A-subunit levels (Hi- and Lo-transgenics, respectively). Transgenics and wild-type littermates were depleted of T regulatory cells (Treg) using antibodies to CD25 (CD4(+) T cells) or CD122 (CD8(+) T cells) before TSHR-adenovirus immunization. Regardless of Treg depletion, high-expressor transgenics remained tolerant to A-subunit-adenovirus immunization (no TSHR antibodies and no hyperthyroidism). Tolerance was broken in low-transgenics, although TSHR antibody levels were lower than in wild-type littermates and no mice became hyperthyroid. Treg depletion before immunization did not significantly alter the TSHR antibody response. However, Treg depletion (particularly CD25) induced thyroid lymphocytic infiltrates in Lo-transgenics with transient or permanent hypothyroidism (low T(4), elevated TSH). Neither thyroid lymphocytic infiltration nor hypothyroidism developed in similarly treated wild-type littermates. Remarkably, lymphocytic infiltration was associated with intermolecular spreading of the TSHR antibody response to other self thyroid antigens, murine thyroid peroxidase and thyroglobulin. These data suggest a role for Treg in the natural progression of hyperthyroid Graves' disease to Hashimoto's thyroiditis and hypothyroidism in humans.

Transgenic mice producing major histocompatibility complex class II molecules on thyroid cells do not develop apparent autoimmune thyroid diseases.

The expression of major histocompatibility complex (MHC) class II molecules on thyrocytes has been demonstrated in autoimmune thyroid diseases. However, the role of this aberrant MHC class II in disease development is controversial. In particular, it remains unknown whether MHC class II expression on thyrocytes, which are nonprofessional antigenpresenting cells, plays a role in inducing autoimmune processes. To clarify this issue, we have produced transgenic mice harboring an MHC class II gene ligated to the promoter of the rat TSH receptor. We obtained three lines of transgenic mice, and the expression of MHC class II by the thyrocytes was demonstrated by immunofluorescence staining and flow cytometry. Our examination revealed no obvious abnormalities in thyroid histology or in thyroid autoantibody production in these transgenic mice. Although serum-free T(4) levels were slightly lower than those of their nontransgenic littermates, no transgenic mouse suffered from clinical hypothyroidism or hyperthyroidism. Furthermore, thyroid lymphocytic infiltration was absent, and MHC class II-expressing thyrocytes obtained from transgenic mice failed to stimulate the proliferation of autologous T cells in vitro. Taken together, these results show that transgenic mice with MHC class II molecules on their thyrocytes do not develop apparent autoimmune thyroid diseases, suggesting that aberrant MHC class II expression alone is not sufficient to induce thyroid autoimmunity.

Coexisting chronic lymphocytic thyroiditis and papillary thyroid cancer revisited.

The effect of chronic lymphocytic thyroiditis (CLT) on the behavior of papillary thyroid cancer (PTC) remains unclear. In recent studies the presence of CLT in patients with PTC was reported to be associated with a lower recurrence rate and an improved survival rate. Furthermore, patients with PTC and tumor infiltrating lymphocytes (TILs) have been reported to have lower recurrence rates and a lower frequency of distant metastases. Because of these and other observations, a tumor immune response in PTC has been suggested. The aim of our study was to determine: (1) the relative frequency of CLT in PTC; (2) the prognostic significance of CLT in patients with PTC; and (3) if TIL occurs independently or in association with CLT. A 10-year retrospective study of patients who underwent initial thyroidectomy for PTC from 1986 to 1996 was completed. The extent of thyroid lymphocytic infiltration was determined within the tumor, surrounding the tumor, and in the distant parenchyma by two independent observers blinded to the clinical data. Dense focal/diffuse lymphoid aggregates throughout the thyroid gland were diagnostic of CLT and when present within or surrounding the tumor were designated TILs. A total of 136 patients with PTC (typical and follicular variant of PTC histologic subtypes) were identified with a mean follow-up of 4.4 years and a 8% mortality rate at 10 years. Thirty percent of the patients with PTC had coexisting CLT, and 65% of these patients with CLT had positive anti-thyroglobulin antibodies. Patients with coexisting CLT and PTC were younger (p < 0.05), more likely to be female (p < 0.05), and more likely to have multicentric tumors (p < 0.001) compared to patients without CLT. Only 5% of patients had TILs without CLT, but 82.5% of patients with CLT had TILs identified (p < 0.0001). By univariate analysis CLT, age, gender, stage of PTC, tumor multicentricity, and tumor size were significant prognostic factors. Only age and TNM stage of PTC remained independent prognostic factors by multivariate analysis. We found a similar frequency (30%) of coexisting CLT and PTC as reported by others; but, more importantly, the presence of TILs primarily occurred in association with CLT. The presence of CLT in patients with PTC correlated with an improved prognosis. It was not an independent prognostic factor, however, and was not associated with a lower recurrence rate or a lower frequency of distant metastasis.

T-Cell Receptors and Autoimmune Thyroid Disease—Signposts for T-Cell-Antigen Driven Diseases

The human autoimmune thyroid diseases (AITDs) are characterized by profuse infiltrates of both CD4+ and CD8+ T cells. The intrathyroidal T-cell-receptor repertoire in Graves' disease, more than in Hashimoto's disease, has been shown to be biased as evidenced by phenotypic analysis and by the use of a restricted T-cell-receptor variable (V) gene repertoire seen in both TCR α and β chains. Evidence for a bias in the T-cell repertoire has also been observed in animal models of induced and spontaneous autoimmune thyroiditis. We found a similar phenomenon of autoimmune thyroid-related T-cell bias in thyroid-humanized scid mice. In these studies we transplanted lymphocyte-depleted thyrocytes and autologous peripheral lymphocytes from AITD patients with a basement membrane preparation which allowed the formation of an artificial thyroid which we have called an “organoid”. T-cell clonal expansion was present in these artificial mixed-cell organoids which appeared to mimic the in vivo process. Such clonal expansion was suggestive of an antigen-driven immune response and could also be identified in thyroid tissue from patients with Graves' disease. Our data on scid mice grafted with human mixed-cell thyroid organoids, therefore, suggested that the major antigens driving T-cell selection in patients with AITD were most likely to be thyroid specific. These antigens include thyroglobulin, thyroid peroxidase, and the receptor for thyroid stimulating hormone (TSHR) on the surface of thyroid epithelial cells and we found significant T-cell proliferation to synthetic TSHR peptides in patients with AITD as compared with normals.Our search for a TCR recognition motif for the autoantigen TPO did not reveal any specific sequence motifs. Instead, analysis of the physico-chemical characteristics i.e. hydrophobicity of the amino acids in the CDR3 (N) region of the TCR α chain, revealed a strong negative linear correlation between strength of stimulation and the average hydrophobicity of N-region amino acids. This led us to hypothesize that lower affinity T-cell clones were commonly more hydrophobic in their CDR3 α region amino acids in keeping with potential crossreactivity of such T cells as a consequence of promiscuous, hydrophobic CDR3 regions. This phenomenon would be analogous to polyreactive, natural autoantibodies which tend to be crossreactive and ‘sticky’. Thus, the physico-chemical characteristics of the TCR α CDR3 region supported the interaction with antigen/MHC by potentially cross-reactive T cells of low affinity.It would seem likely that such low-affinity autoreactive T-cell populations serve as a pool of potentially pathogenetic cells. These cells would be able to respond to an insult which, via a number of posssible mechanisms such as molecular mimicry, would initiate a thyroid lymphocytic infiltration in an antigen-driven fashion with intrathyroidal T-cell expansion and a marked bias in the utilization of T-cell-receptor V genes.

Fas/Fas ligand up-regulation and Bcl-2 down-regulation may be significant in the pathogenesis of Hashimoto's thyroiditis.

Hashimoto's thyroiditis (HT) is an autoimmune disorder characterized by diffuse thyroid lymphocytic infiltration and follicle destruction. Cross-linking of the Fas receptor with its own ligand (FasL) triggers apoptosis in various systems, whereas the Bcl-2 protooncogene inhibits apoptotic cell death. The involvement of Fas, FasL, and Bcl-2 in the apoptotic process in HT was evaluated in 15 thyroid tissue samples from patients with HT stained for apoptosis and for Fas, FasL, and Bcl-2 protein expression. Eight samples from healthy thyroid tissue were used for comparison. Thyroid follicles in HT samples exhibited strong staining for Fas and FasL and a high percentage of apoptosis (30.3 +/- 14.5%, mean +/- SD), in contrast to normal control follicles that exhibited moderate Fas, minimal or no FasL, and hardly any apoptosis. Immunostaining for Bcl-2 was high in normal, and weak in involved, thyroid follicles. Infiltrating lymphocytes stained weakly for FasL and strongly for Bcl-2. We conclude that follicular cells in HT undergo apoptosis by concomitant up-regulation of FasL and Fas and down-regulation of Bcl-2 protein. The lymphocytes do not seem to be directly engaged in the process with their own FasL, but they may provide the appropriate cytokine milieu that, in turn, up-regulates Fas and/or FasL leading to apoptosis.