Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Mar 1.
Published in final edited form as: Curr Protoc. 2024 Mar;4(3):e938. doi: 10.1002/cpz1.938

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

Aizhan Kozhakhmetova 1,*, Yaron Tomer 1,*, Mihaela Stefan-Lifshitz 1,†
PMCID: PMC10917119  NIHMSID: NIHMS1947327  PMID: 38436133

Abstract

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 since 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. We describe a protocol for induction of EAT by immunization of mice susceptible to thyroiditis with an adenovirus vector carrying full-length human TG cDNA (Ad-TG EAT). We provide supporting 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 of 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).

BASIC PROTOCOL 1: 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), 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)

Keywords: thyroglobulin, genetic variants, cDNA immunization, adenovirus, experimental autoimmune thyroiditis

INTRODUCTION

Autoimmune thyroid diseases (AITD) including Graves’ disease (GD) and Hashimoto’s thyroiditis (HT) are common autoimmune disorders that occur when a combination of genetic susceptibility variants and environmental factors leads to loss of immune self-tolerance at the central and peripheral levels (1). Thyroglobulin (TG), a 660-kDa glycoprotein that represents 75–80% of the total thyroid protein content, constitutes an important target of the autoimmune response in AITD, and it has been recognized as the key autoantigen in thyroid autoimmunity (2). Indeed, anti-TG antibodies (Abs) are present in 75% of the AITD patients (3), and in a mouse model of spontaneous autoimmune thyroiditis breakdown of self-tolerance happens first for TG and subsequently for thyroid peroxidase (TPO) (4). Moreover, the TG gene has emerged as a key major AITD susceptibility gene in whole-genome linkage and association studies (5–13). Several non-synonymous single nucleotide polymorphisms (SNPs) in the TG coding region were identified to be strongly associated with AITD (14, 15). Additionally, gene-gene interactions that significantly increase the odds ratio (OR) for GD have been found between a TG exon 33 SNP and an HLA-DRβ1 variant, HLA-DRβ1-Arg74 (14, 16).

To understand the functional significance of the TG coding variants, their interactions with other AITD-susceptible gene variants (e.g., HLA-DRβ1-Arg74) as well as with environmental factors, we developed a mouse model of experimental autoimmune thyroiditis (EAT) by using immunization with an adenovirus vector carrying full-length cDNA of human TG (Ad-TG) (17). Compared with the classical EAT model, which is based on immunizing genetically susceptible mice with TG protein in adjuvant (e.g., complete Freund’s adjuvant – CFA or bacterial lipopolysaccharide - LPS) (18), the Ad-TG EAT mouse model present unique advantages for genetic functional studies. Manipulation of the TG cDNA, by oligonucleotide-directed mutagenesis or DNA synthesis to introduce variants associated with AITD, allows testing of the functional effects of susceptible variants and their haplotypes on TG immunogenicity and EAT development. Furthermore, the Ad-TG EAT mouse model is suitable for studying the interactions of the susceptible TG variants with factors known to impact AITD development (e.g., cytokines, iodine intake), or with variants of other AITD susceptible genes, particularly, HLA-DRβ1-Arg74 (e.g., by Ad-TG immunization of NOD mice expressing human HLA-DRβ1-Arg74 (19, 20)).

We describe here the protocol for induction and evaluation of Ad-TG EAT in mouse strains susceptible to autoimmune thyroiditis. EAT is assessed by measuring in vitro T cell proliferative responses to TG and to immunogenic peptides derived from TG (Support Protocol 2) and cytokines secretion (Support Protocol 3) as well as by evaluating thyroid histology (Support Protocol 4) and thyroid lymphocytic infiltration by immunohistochemistry (Support Protocol 5). We also describe measurement of anti-TG antibodies in mouse sera (Support Protocol 6).

BASIC PROTOCOL 1: Development of a mouse model of autoimmune thyroiditis induced by immunization with adenovirus containing full-length thyroglobulin cDNA.

Human thyroglobulin (hTG) is a large complex gene spanning 270 Kb on chromosome 8q24 comprising 48 exons (mRNA NCBI Reference Sequence: NM_003235); the full-length TG cDNA encompasses 8,450 Kb. Thus, studies with full-length TG cDNA can pose technical challenges during sequencing, cloning, and manipulations. To generate the Ad-TG construct the full-length hTG cDNA is assembled from synthetic oligonucleotides and inserted into a plasmid. Mutations can be introduced into TG cDNA in this step by oligonucleotide-directed mutagenesis or DNA synthesis. The TG cDNA is then incorporated into adeno-associated virus (AAV) vector (Vector BioLabs, Malvern, PA).

Materials:

Mouse stains:

CBA/J - 6–8 weeks old, Females (Jackson Laboratory)

NOD.H-2h4 – 8-10 weeks old, Males and Females

Reagents:

  • hTG cDNA packaged into an adenovirus vector (Ad-TG) (Vector BioLabs)

  • LacZ cDNA packaged into an adenovirus vector (Ad-LacZ) (Vector BioLabs)

  • Phosphate-buffered saline (PBS) (ThermoFisher, cat. no. J61196.AP)

  • 70% ethanol (ThermoFisher, cat. No. T038181000)

  • 10% glycerol in PBS (see Reagents and Solutions)

  • Formalin fixative: 10% formalin in 0.15 M sodium acetate

Equipment:

  • ✓ Hamilton glass syringe (Hamilton, cat. No. 81320)

  • ✓ Insulin syringe (0.5 ml) (BD, cat. No. 329461)

  • ✓ 18-G (BD, cat. no. 305196) and 25-G needles (BD, cat. no. 305122)

  • ✓ Sharp and round forceps (Roboz Surgical Instruments)

  • ✓ Scalpel

  • ✓ Scissors

  • ✓ 50 ml conical tubes

  • ✓ Histology sample cassettes

  • ✓ 1.5 ml tubes

Protocol steps:

Immunization of mice with adenovirus expressing hTG.

  • 1

    Keep the mice in a controlled environment with 12-hour light-dark cycles (starting at 7 AM and 7 PM) and a room temperature (RT) of 22°C. Ensure the mice have unrestricted access to standard rodent chow and water.

  • 2

    Immunize mice aged 6 – 10 weeks with Ad-hTG or Ad-LacZ. Inject intramuscularly with 5.0 × 109 particles of adenoviral vector containing full-length hTG (Ad-TG) (experimental mice) or control LacZ (Ad-LacZ) cDNA (control mice) in 50 μl of 10% glycerol (in PBS), in the thigh muscle on days 0, 21, and 42. Typically we use at least 5 experimental and 5 control mice for the experiment. Development of thyroiditis is evaluated only after 62 days. Although not expected, mice are checked twice per week for signs of distress or abnormal behavior.

  • 3

    Euthanize mice on day 63 (Figure 1).

Figure 1. Immunization protocol with adenovirus containing full-length thyroglobulin cDNA.

Figure 1.

Open in a new tab

Mice are injected intramuscularly (IM) with 5.0 × 109 particles of adenoviral vector containing full-length hTG (Ad-TG) or control LacZ (Ad-LacZ) cDNA, 50 μl (in BPS), in the thigh muscle. A series of three intramuscular injections is given on day 0, day 21, and day 42 and mice are euthanized on day 63.

Dissection: organ harvesting and blood collection.

  • 4

    Clean dissection area with 70% ethanol.

  • 5

    Expose chest cavity and collect the whole blood directly from heart using insulin syringe into a 1.5 ml tube (Figure 2).

  • 6

    Expose abdominal cavity, remove spleen from mice cutting out with small scissors, put in 15 ml cold PBS solution. Isolated splenocytes will be used for cell proliferation assay and cytokine assay in detailed in Support Protocols 1–3.

  • 7

    Surgically access the larynx and trachea by peeling back the neck muscles. Utilize small scissors to create incisions, positioned above the larynx and in the middle section of the trachea. Remove the larynx and trachea along with attached thyroid lobes.

  • 8

    Place the thyroid biospecimen in a histology sample cassette and label it with sample number. Fix the tissues by putting in 10% formalin fixative for 24h, then keep in 70% ethanol for the hematoxylin and eosin (H&E) staining (Support Protocol 4), as well as immunohistochemistry (Support Protocol 5).

  • 9

    Allow blood to clot at RT for 20 – 30 min. Spin down the blood at 7,000 rpm for 10 min. After serum is separated from the clot, collect sera for further measurements of serum total thyroxine (tT4) (not described) as well as anti-mouse thyroglobulin (mTg) antibodies using ELISA (Support Protocol 6). Store serum samples at - 20°C.

Figure 2. Mouse dissection, organ harvesting and blood collection.

Figure 2.

Open in a new tab

(A) Blood collection: clean dissection area with 70% ethanol. Make an incision on the skin and cut through the sternum and spread the ribs to expose the heart. Utilize a 0.5 ml insulin syringe to extract blood from the heart by pinching it with the needle and aspirating the blood into the syringe. Transfer the collected blood into a 1.5 ml tube. (B) Spleen harvesting expose the abdomen by making a vertical incision on the skin and proceed to separate the spleen. Place the dissected spleen into a tube containing 15 ml of ice-cold PBS. (C) Thyroid harvesting: create a vertical cut in the neck area, separate the neck muscles to reveal the larynx and trachea. Incise the fascia above the trachea and gently lift it upward utilizing forceps. With the curved scissors carefully separate trachea from the surrounding soft tissues and cut the larynx above the thyroid and then cut below the forceps in the middle section of trachea, extract the larynx and upper trachea along with the attached thyroid lobes and put into a histology sample cassette.

SUPPORT PROTOCOL 1. Splenocytes isolation

In this protocol we describe how to isolate splenocytes from mouse spleen.

Materials:

Reagents:

  • Complete RPMI medium: 500 ml RPMI w/glutamine (Corning, cat. no. 10040CV) + 10% FBS (Sigma-Aldrich, cat. no. 12007C) +1% Sodium pyruvate (Sigma-Aldrich, cat. no. S8636) (see Reagents and Solutions)

  • Phosphate-buffered saline (PBS) (ThermoFisher, cat. no. J61196.AP)

  • Ammonium-chloride-potassium buffer (4g NH4Cl + 0.5g KHCO3 + 100μlEDTA + up to 500 ml distilled H2O, pH 7.2–7.4) (see Reagents and Solutions)

Equipment:

  • ✓ Cell strainers (100 μm) (Falcon, cat. no. 352360)

  • ✓ Tissue culture dish (100 × 20 mm) (Falcon, cat. no. 353003)

  • ✓ Syringe (10 ml) (BD, cat. no. 302995)

  • ✓ Insulin syringe (0.5 ml) (BD, cat. No. 329461)

  • ✓ 15 ml conical tube (Falcon, cat. no. 352196)

Protocol steps:

  1. Use the mouse spleen dissected in Basic Protocol 1 step 6. Put it in complete RPMI 1640 medium.

  2. Put a cell strainer on top of a 50 ml falcon tube.

  3. Place the spleen in the cell strainer, use a 10-ml syringe plunger to press the spleen (use circular motion).

  4. Filter the suspension through a 100 μm cell strainer twice and centrifuge at 200 g for 10 minutes at 4°C.

  5. Wash the pellet with complete RPMI followed by centrifugation at 200g for 10 minutes at 4°C.

  6. To remove red blood cells, add 5 ml of Ammonium-Chloride-Potassium (ACK) lysis buffer to the cell pellet.

  7. Incubate cells with ACK lysis buffer for 5 min at room temperature while intermittently shaking, then add 10 ml of complete RPMI, centrifuge at 200 g for 10 min at 4°C and discard supernatant.

  8. Resuspend the remaining pellet in 10 ml of complete RPMI, filter the suspension through a 100 μm cell strainer, centrifuge at 200 g for 10 minutes at 4°C and discard supernatant.

  9. Resuspend the cell pellet in 3 ml of complete RPMI and keep on ice.

Count cells and use them for further experiments. Usually we get 50–60 mln splenocytes per a mouse spleen, but the amount can vary.

SUPPORT PROTOCOL 2: T cell stimulation and carboxyfluorescein diacetate succinimidyl ester (CFSE) based cell proliferation assay

The carboxyfluorescein diacetate succinimidyl ester (CFSE) based cell proliferation assay is a widely used technique to assess the division of cells in vitro. The assay relies on the covalent attachment of the CFSE fluorescent dye to intracellular proteins of live cells. Upon cell division, the dye is evenly distributed between the daughter cells, resulting in the halving of fluorescence intensity in each generation. This feature allows for the monitoring of the number of divisions that cells undergo with flow cytometry. Adding different stimulants to the CFSE-labelled cells we can assess the effect on their activation and proliferation.

Materials:

Reagents:

  • Isolated splenocytes from Ad-TG and Ad-LacZ immunized mice (support protocol 1 step 9)

  • Carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen, cat. no. V12883)

  • Phosphate-buffered saline (PBS) (ThermoFisher, cat. no. J61196.AP)

  • 0.1% bovine serum albumin (BSA) in PBS (see Reagents and Solutions)

  • Complete RPMI medium: 500 ml RPMI w/glutamine (Corning, cat. no. 10040CV) + 10% FBS (Sigma-Aldrich, cat. no. 12007C) +1% Sodium pyruvate (Sigma-Aldrich, cat. no. S8636) (see Reagents and Solutions)

  • Negative control peptide (GenScript)

  • Dynabeads Mouse T-activator CD3/CD28 (Gibco, cat. no. 11452D)

  • Human thyroglobulin (hTG) (Cell Science, cat. no. CSI14829B)

  • Human thyroglobulin peptides (TG2098, TG202, TG726, TG1951, and TG1571) (GenScript)

Equipment:

  • ✓ Round-bottom 96-well cell plate (Corning, cat. no. 3799)

  • ✓ 50 ml conical tubes (ThermoScientific, cat. no. 339652)

  • ✓ Multichannel pipette

  • ✓ 1.5 ml tubes

  • ✓ Flow cytometer BD Accuri C6 (BD Biosciences)

  • ✓ Flowjo software (Tree Star)

Protocol steps:

  1. Allow CFSE and DMSO to warm to room temperature (RT). Dissolve content of CFSE vial in 90 μl of DMSO to obtain 10mM CFSE solution.

    Make 15 μl aliquots and store up to 1 year at - 20°C.

  2. Dilute 10 μl of 10 mM CFSE in 10 μl of PBS to obtain 5 mM CFSE solution.

  3. Dissolve 5 mM CFSE in 0.1% BSA/PBS to obtain a 3 μM (2x) concentration of CFSE mix well.

    Prepare CFSE working solution just prior to adding to the cells.

  4. Count splenocytes and resuspend 2 × 106 cells in 10 ml of 0.1% BSA in PBS.

  5. Split cell suspension into two new 50 ml Falcon tubes (5 ml with 1 × 106 cells in each).

  6. Add 5 ml of 3 μM CFSE to each tube. Mix by inverting, incubate for 10 min at 37°C.

  7. Terminate the staining by adding 40 ml of ice-cold complete RPMI medium to each tube, leave 5 min on ice, then centrifuge at 1,500 rpm for 10 min, discard supernatant.

  8. Wash cells with 10 ml of medium at RT 3 times.

  9. Combine the cell pellet from both tubes by resuspending in 3 ml of fresh medium. Count cells.

  10. Plate 100 μl (2 × 105 cells/well) of CFSE-labeled cells on 96-well plate.

  11. Treat the cells in different wells with (1) medium; (2) negative control peptide, 20 μg/ml; (3) mouse CD3/CD28 beads (as positive control), 5 μl per 106 cells; (4) hTG, 40 μg/ml, or (5) five hTG peptides (TG.2098, TG.202, TG.726, TG.1951, and TG.1571) (20), each peptide at 20 μg/ml (see peptide sequencies in Table 1). Make each condition in quadruplicate. Incubate at 37°C in 4%-CO2 incubator for 5 days.

  12. After 5 days, pipette out the cells from the 96-well plate into flow tubes with 650 μl of PBS in each, spin at 1,200 rpm, 10 min at RT, discard supernatant.

  13. Add 300 μl of PBS into each tube.

  14. Analyze T cell proliferation by flow cytometry analysis, utilizing Flowjo software. Express data as stimulation index. Calculate the stimulation index by using the following formula: Stimulation index = [% proliferating lymphocytes (treated)] / [% proliferating lymphocytes (not treated)].

Table 1.

Amino acid sequencies of peptides used for T cells stimulation in the CFSE cell proliferation assay (Support Protocol 2).

Peptide Amino acid sequence
TG.2098 LSSVVVDPSIRHFDV
TG.202 VNTTDMMIFDLVHSYNRFPD
TG.726 CPTPCQLQAEQAFLRTV
TG.1951 FRKKVILEDKVKNF
TG.1571 EKVPESKVIFDANAPVAVRSKVPDSEF
Negative control peptide PKDRLKIYNNFTKIGDLSL
Open in a new tab

As a result of the assay, we observed that mice immunized with Ad-TG developed significantly higher T cell proliferative responses to hTG and TG.2098, when compared to response to medium, while mice immunized with AD-LacZ did not (17).

SUPPORT PROTOCOL 3: Cytokine assays: measuring levels of interferon gamma (IFNγ), interleukins (IL)-2, IL-4 and IL-10 in splenocyte supernatants.

The aim of the assay is to assess T-cell responses to thyroglobulin and immunogenic thyroglobulin peptides (see Table 1) by measuring cytokines produced by activated T-cells.

Materials:

  • Isolated splenocytes from Ad-TG and Ad-LacZ immunized mice

  • Milliplex mouse cytokines/chemokine magnetic panel (MCYTOMAG-70K kit for INFγ, IL2, IL4, IL10 measurement) (EMD Millipore Corporation)

    Kit includes 25-plex antibody-beads (1 bottle), 32-plex antibody-beads (1 bottle), standard (1 l/vial), quality controls 1 and 2 (2 l/vials), serum matrix (1 l/vial), assay buffer (1 bottle), 10X wash buffer (1 bottle), mouse cytokine antibodies (1 bottle), streptavidin-phycoerythrin (1 bottle), mixing bottle (empty), 96-well Opti plate.

  • Sheath fluid (15 ml, from Luminex supply)

  • Phosphate-buffered saline (PBS)

  • 0.1% bovine serum albumin in PBS

  • Complete RPMI 1640 medium (RPMI 1640/10% FBS/1% Sodium pyruvate)

  • ddH20

  • Human thyroglobulin (hTG) (Cell Science, cat. no. CSI14829B)

  • Human TG peptides (TG2098, TG202, TG726, TG1951, and TG1571) (GenScript) (Table 1)

  • Negative control peptide (Table 1) (GenScript)

  • Dynabeads Mouse T-activator CD3/CD28 (Gibco, cat. no. 11452D)

Equipment:

  • ✓ Round-bottom 96-well cell plate (Corning, cat. no. 3799)

  • ✓ 50 ml conical tubes (ThermoScientific, cat. no. 339652)

  • ✓ Reagent Reservoir (ImmunoWare, cat. no. 15075)

  • ✓ Aluminum Foil (Fisherbrand, cat. no. 15-078-292)

  • ✓ 8-channel pipette, 30–300 μl (Eppendorf, cat. no. FS-0098499)

  • ✓ 1.5 ml tubes

  • ✓ Graduated cylinder

  • ✓ Titer plate shaker (VWR, cat. no. 12620–926)

  • ✓ Ultrasonic cleaner (Branson model B200) (Fisher, cat. no. 1533722)

  • ✓ Luminex 200 plate reader with xPONENT software (Luminex)

Protocol steps:

Stimulation of splenocytes:

  • 1

    Count isolated splenocytes from mice (see support protocol 1 step 9) and dilute to obtain concentration of 2 × 106 cells/ml.

  • 2

    Plate 100 μl (2 × 105 cells/well) of cells in a 96-well plate.

  • 3

    Treat the cells with: (1) medium (100 μl); (2) negative control peptide, 20 μg/ml; (3) mouse CD3/CD28 beads (as positive control), 5 μl/106 cells; (4) TG, 40 μg/ml; and (5) five hTG peptides (TG2098, TG202, TG726, TG1951, and TG1571), 20 μg/ml. Make each condition in quadruplicate. Incubate at 37°C in 4%-CO2 incubator for 48 h.

  • 4

    After incubation, collect supernatants from wells into tubes, spin at 1,500 rpm, 10 min at 4°C. Collect the supernatant.

  • 5

    Measure the cytokine/chemokine levels by using a Milliplex mouse cytokines/chemokine magnetic panel (catalog no. MCYTOMAG70K) following the manufacturer’s instructions described in further steps (steps 6–20)

Preparation of Reagents for Immunoassay

  • 6

    Preparation of Antibody-Immobilized Beads: sonicate each vial antibody bead for 30 seconds; vortex for 1 minute. Add 150 μl from each antibody bead vial to the Mixing Bottle and bring final volume to 3.0 ml with Bead Diluent, mix well. Unused portions may be stored at 2–8°C for up to one month.

    Due to the composition of magnetic beads, you may notice a slight color in the bead solution. This does not affect the performance of the beads or the kit.

  • 7

    Preparation of Quality Controls: reconstitute Quality Control 1 and Quality Control 2 with 250 μl deionized water, invert the vials several times to mix and vortex. Leave the vials for 5–10 minutes on the bench. Transfer the reconstituted Quality Control 1 and Quality Control 2 into two polypropylene microfuge tubes. Unused portion may be stored at - 20°C for up to one month.

  • 8

    Preparation of Wash Buffer: warm the 10X Wash Buffer to room temperature (RT) and mix to bring all salts into solution. Dilute 50 ml of 10X Wash Buffer with 450 ml deionized water to get 1X concentration. Store unused portion at 2–8°C for up to one month. Add 200 μl of 1X Wash Buffer to each plate well, seal plate, and put on shaker for 10min at 700 rpm.

  • 9

    Mouse Cytokine Standard preparation: reconstitute Mouse Cytokine Standard with 250 μl ddH2O – this will be Standard 6. Mix by inverting, vortex for 10sec, let vial sit at RT for 5–10min. Label 5 microfuge tubes as Standards no. 1–5. Add 200 μl of Assay Buffer to each of 5 tubes. Prepare serial dilutions: vortex and add 50 μl of Standard 6 to the Standard 5 tube, vortex, transfer 50 μl of Standard 5 to Standard 4 tube, vortex, transfer 50 μl of Standard 4 to Standard 3 tube, vortex, transfer 50 μl of Standard 3 to Standard 2 tube, vortex, transfer 50 μl of Standard 2 to Standard 1 tube, vortex.

Immunoassay Procedure

  • 10

    Decant wash buffer from shaken plate into sink and tap on a paper towel to fully dry. Add 25 μl of each Standard or Control into the appropriate wells according to the Plate map. Background (Standard 0) should be filled with 25 μl of Assay Buffer.

  • 11

    Add 25 μl of Assay Buffer to the Sample wells (not Standard or Control wells).

  • 12

    Add 25 μl of matrix solution (cell culture medium) to Background, Standard, and Control wells (not Sample wells).

  • 13

    Add 25 μl of Samples 1–39 to appropriate wells.

  • 14

    Vortex Mixing Bottle and add 25 μl of Mixed Beads to all wells (shake bottle often). Seal plate, wrap with aluminum foil, and incubate on a plate shaker overnight at 4°C (or for 2 hours at RT).

  • 15
    After completing this step, wash plate 2 times:
    1. Place plate on magnet, lock, and let sit for 60 sec so beads can settle.
    2. While still holding plate to magnet, dump out plate in waste bucket, tap on paper towels.
    3. Add 200 μl of 1X Wash Buffer to all wells, remove from magnet, shake for 30 sec 700 rpm at RT. Repeat steps a-c for a second wash. Repeat steps a-b to remove well contents.
  • 16

    Add 25 μl of Detection Antibodies to all wells, seal, cover with aluminum foil, incubate on shaker for 1hr, 700 rpm at RT.

  • 17

    Add 25 μl Streptavidin-Phycoerythrin to all wells, seal with adhesive film, foil, incubate on shaker for 30 min at RT.

  • 18

    Wash plate 2 times with 200 μl of 1X Wash Buffer (follow step 6 above).

  • 19

    Add 150 μl of Sheath Fluid to all wells, put the plate on a plate shaker for 5 minutes to resuspend the beads.

  • 20

    Run plate on Luminex® 200™. Save and analyze the Median Fluorescent Intensity (MFI) data using a 5-parameter logistic or spline curve-fitting method for calculating analyte concentrations in samples.

In this experiment we usually measure IFNγ, IL-2, IL-4, IL-10 and IL-17 cytokines. We found that immunization of mice with Ad-TG triggered increased secretion of Th1-type cytokines including IFNγ and IL-2 ((17).

SUPPORT PROTOCOL 4: Evaluating thyroid histology and infiltration with immune cells: hematoxylin-eosin staining of mice thyroid glands.

Hematoxylin-eosin (H&E) staining is a widely used histological technique to examine the morphology and structure of tissues under a microscope. In thyroid gland, H&E staining can reveal important information about the cellular composition (e.g., lymphocytic infiltration) and architecture of the tissue. By analyzing the H&E-stained sections of the thyroid, we can recognize thyroid morphology as well as lymphocytic infiltration of the thyroid.

Materials:

Reagents:

  • Thyroid tissues fixed in 10% neutral-buffered formalin and embedded in paraffin

  • Xylene (ThermoFisher, cat. no. 447240010)

  • 80%, 95%, 100% ethanol (ThermoFisher, cat. no. T0381810000)

  • Acid ethanol: 1 ml concentrated HCl + 400 ml 70% ethanol

  • Hematoxylin (Poly Scientific, cat. no. s212A)

  • Eosin (Poly Scientific, cat. no. s176)

  • Histological mounting medium Permount (Fisher, cat. no. SP15–100)

  • Deionised water

Equipment:

  • ✓ Glass coverslips

  • ✓ Coplin jars

  • ✓ Xylene resistant forceps for handling slides

  • ✓ Upright microscope or 3DHistec Panoramic 250 Flash II slide scanner

Protocol steps:

  1. Fix the thyroids (from basic protocol 1 step 8) in 10% neutral-buffered formalin and then embed them in paraffin.

  2. Prepare sections (4 um thick) for staining with H&E.

  3. In a slide holder (glass or metal), place slides containing paraffin sections.

  4. Dehydrate and rehydrate sections: blot excess xylene before proceeding to ethanol, perform 3 × 3 min dips into xylene, 3 × 3 min into 100% ethanol, 1 × 3 min into 95% ethanol, 1 × 3 min into 80% ethanol, and 1 × 5 min into deionized H2O.

  5. Clean the surface of hematoxylin with a kimwipe to remove oxidized particles while the sections are in water. Before immersing in hematoxylin, blot excess water from the slide holder.

  6. Stain with hematoxylin for 1 × 3 min, then rinse with deionized water and 1 × 5 min with tap water to allow the stain to develop.

  7. Dip into acid ethanol 8–12 times quickly for destaining. Rinse twice for 1 min with tap water, followed by a 2 min rinse with deionized water (at this stage, it can be left overnight). Before proceeding to eosin, blot excess water from the slide holder.

  8. Perform eosin staining and dehydration: 1 × 30 sec dip into Eosin, 3 × 5 min into 95% ethanol, and 3 × 5 min into 100% ethanol. Blot excess ethanol before immersing in xylene. Perform 3 × 15 min dip into xylene.

  9. Using a glass rod, place one drop of permount on the slide, ensuring no bubbles are present. Tilt the coverslip and gently let it fall onto the slide. Allow the permount to spread beneath the coverslip, covering all the tissue. Dry the slide overnight in the hood.

  10. Acquire the images by using a microscope or a slide scanner [3DHistec Panoramic 250 Flash II slide scanner (Figure 3)].

Figure 3. Hematoxylin and eosin (H&E)-stained sections of thyroid from mice immunized with Ad-TG and Ad-LacZ.

Figure 3.

Open in a new tab

(A) Thyroid section of control mice (Ad-LacZ). (B) Lymphocytic infiltration in thyroid of Ad-TG immunized mice (Ad-TG). Images were obtained using slide scanner 3DHistec Panoramic 250 Flash II slide scanner and analyzed using CaseViewer (10x).

SUPPORT PROTOCOL 5: Immunohistochemistry of thyroid tissues: Immunofluorescence protocol of paraffin-embedded thyroid sections.

Immunohistochemistry (IHC) of thyroid tissues is a technique used to detect and visualize proteins or antigens that are specific to certain cell types within the tissue using labeled antibodies. In the thyroid gland IHC is useful for identifying specific cell types and characterizing their distribution within the tissue. It can be used to identify and visualize immune cells within the thyroid gland by using labeled antibodies that target surface molecules specific to certain immune cells.

Materials:

Reagents:

  • Xylene (ThermoFisher, cat. no. 447240010)

  • Ethanol 70–100% (ThermoFisher, cat. no. T038181000)

  • Phosphate-buffered saline (ThermoFisher, cat. no. J61196.AP)

  • Target Retrieval Solution (DAKO, cat. no. S1699)

  • Pap pen (DAKO, cat. no. S2002)

  • Blocking serum (10% BSA/TRIS 1X/0.1% Triton)

  • DAPI (4’,6-diamidino-2-phenylindole) (Sigma, cat. no. 28718-90-3)

  • Fluoromount-G (Southern Biotech, cat. no. 0100–01)

  • Primary antibody: Anti-CD45 Rat Anti-Mouse Antibody 1:100 (Life Technologies, cat. no. 14-0451-85); Anti-B220 Rat Anti-Mouse Antibody 1:100 (BD Biosciences, cat. no. 550286); Anti-CD3 Rabbit Antibody 1:200 (Abcam, cat. no. 16669–200); Anti-CD4 Rabbit Antibody 1:200 (Cell Signaling, cat. no. 25229)

  • Secondary antibody: Goat Anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluortm 568 1:500 (Thermofisher, cat. no. A-11004); Goat Anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluortm 488 1:500 (Thermofisher, cat. no. 32731).

Equipment:

  • ✓ Coplin jar

  • ✓ Coverslips (Fisherbrand, cat. no. 12–545-F)

Protocol steps:

  1. Rehydrate thyroid sections:
    1. Immerse the tissue in Xylene, 2 times for 5 minutes each,
    2. Immerse the tissue in ethanol 100% - 20 dips,
    3. Immerse the tissue in ethanol 90% - 20 dips,
    4. Immerse the tissue in ethanol 80% - 20 dips,
    5. Immerse the tissue in ethanol 70% - 20 dips,
    6. Rehydrate with Phosphate-buffered saline (PBS), 2 times for 10 min each.

  2. Prepare antigen retrieval solution: dilute Target Retrieval Solution 1/10 in distilled water in a plastic coplin jar suitable for the pressure cooker.

  3. Place the coplin jar inside the pressure cooker. Set the pressure cooker for 15 min at High pressure.

    Remove the pressure after 15 min.

  4. After 15 minutes take the coplin jar from pressure cooker and wash with PBS 2 times for 5 min (quick washes).

  5. Continue with the immunohistochemical staining steps below:
    1. Dry edges of slides with paper towel.
    2. Circle sections with Pap pen, never allow sections to dry during the procedure.
    3. Block the slides with blocking serum for 30 – 45 min.
    4. Add primary antibody diluted in blocking serum and incubate in humid chamber for 60 min at room temperature (RT) or overnight at 4°C (cover completely the section 15–50 μl is enough dependent on the tissue). For thyroid tissues (3–4 sections), incubate with 100 μl of primary antibodies.
    5. Dry out the excess of primary antibodies.
    6. Wash the slides 3 times for 5 minutes in 1X PBS (dip multiple times in PBS).
      Leave slides in PBS while preparing secondary antibodies.
    7. Add secondary antibody diluted in PBS and incubate at RT in humid chamber for 30 min (cover completely the section, 20–80 μl is enough).
      Add 100–200 μl depending on how many sections. Cover with aluminum foil.
    8. Wash off secondary antibody with PBS 3–4 times. Dip into PBS multiple times in every wash.
    9. Stain with DAPI (1 μg/ml in PBS) for 5 min at RT in the dark.
      Leave slides immersed in DAPI instead of adding it directly to coverslips.
    10. Wash 1 time in 1X PBS.
      Just rinse in PBS before mounting.
    11. Take the slides from the coplin jar, and without letting them dry cover the sections with a drop of Fluoromont-G and with a coverslip.
    12. Wait until the Fluoromont-G is completely dry.
    13. Check staining under the microscope.
    14. Seal edges with coverslip sealant.
      This step is necessary if an inverted microscope is used.
    15. Take photos on the following day after Fluoromont-G is dried overnight (Figures 4 – 7).

Figure 4. Immunostaining of CD45+ T-cells in thyroid sections from Ad-TG and Ad-LacZ immunized NOD.H-2h4 mice.

Figure 4.

Open in a new tab

(A) Immunostaining shows very few CD45 positive cells in thyroid of control mice (Ad-LacZ). (B, C) Abundant CD45+ (red) T-cells in thyroid infiltrates of Ad-TG immunized mice (Ad-TG). Images were taken using a confocal microscope (A and B – 20x, C – 40x). Red, CD45+ T-cells staining; Blue, DAPI staining for nuclei.

Figure 7. Immunostaining for B220 + B-cells of thyroid sections from Ad-TG and Ad-LacZ immunized NOD.H-2h4 mice.

Figure 7.

Open in a new tab

(A) No B220+ B-cells are present in the thyroid of control mice (Ad-LacZ). (B) Clusters of B220+ B-cells (red) are seen in areas of the thyroid infiltrates of Ad-TG immunized mice (Ad-TG). Images were taken using a confocal microscope (A and B – 20x). Red, staining for B202+ B-cells; Blue, DAPI staining for nuclei.

SUPPORT PROTOCOL 6: Anti-thyroglobulin antibody measurement in mice sera by enzyme-linked immunosorbent assay (ELISA)

Anti-thyroglobulin antibodies (mTg Abs) are specific antibodies that can be detected in autoimmune thyroiditis. In this protocol we will describe measurement of mTg Abs utilizing enzyme-linked immunosorbent assay (ELISA) in mice sera.

Materials:

Reagents:

  • Sera from mice induced with EAT

  • Easy Sigma Fast Para-nitrophenylphosphate tablets (PNPP) (Sigma, cat no. N-1891)

  • Mouse Tg (mTg) (stock 4 mg/ml)

  • Carbonate-Bicarbonate buffer p.h.9.6 (Fisher, cat. no. 50-100-88) (dissolve 1 capsule in 100 ml ddH2O, pH = 9.6) (See Reagents and Solutions)

  • anti-mouse IgG secondary antibody (Sigma, cat no. A5324)

  • Phosphate-buffered saline (ThermoFisher, cat. no. J61196.AP)

  • Bovine serum albumin (BSA) (Sigma, A3294–10G)

  • Tween-20 (Fisher, cat. no. 28320)

  • ddH2O

  • Buffers:
    1. 0.5% BSA/PBS: add 0.05g BSA to 10 ml of PBS – sera dilution buffer,
    2. 0.05% PBS-Tween (PBST): 500 ml PBS + 250 μl Tween - washing buffer,
    3. 2.5% BSA in PBST: for one plate, to 20 ml PBST add 0.5g of BSA - blocking solution,
    4. 1% BSA/PBST: add 0.3g of BSA to 30 ml of PBST – secondary antibody dilution buffer.

Equipment:

  • ✓ 96-well plate (Dynatech Immulon) VWR, cat. no. 62402–952)

  • ✓ Reagent Reservoir (ImmunoWare, cat. no. 15075)

  • ✓ Multichannel pipettes

Protocol steps:

  1. Prepare 10 μg/ml of mTg in bicarbonate buffer from stock mTg (4 mg/ml).

  2. Coat ELISA 96-well plate with 100 μl of 10 μg/ml of mTG per well. Cover with parafilm. Incubate 4°C overnight.

  3. On next day, remove the plate coating by using multichannel pipette, wash plate with PBST 4 times.

  4. Add 200 μl blocking solution (2.5% BSA/PBST) to each well using multichannel pipette, cover with film and incubate for 60 min at 37°C in dry air incubator.

  5. Vortex and dilute test sera, positive and negative controls 1:100 (4 μl of serum in 396 μl of 0.5% BSA/PBS). Vortex and keep on ice.

  6. Wash plate with PBST 6 times. Use a vacuum to dry out if necessary.

  7. Add 100 μl/well of diluted sera and controls in triplicates. Blank – 0.5% BSA/PBS. Incubate 2 h at room temperature (RT) with slow shaking (~150 rpm).

  8. Wash unbound sera with PBST 6 times.

  9. Dilute anti-mouse IgG secondary antibody 1:30,000 in 1% BSA/PBST. Add 100 μl of diluted secondary Ab to each well. Incubate 37°C 30 min in dry air incubator.

  10. Wash off secondary Ab with PBST 4 times.

  11. Add 100 μl freshly prepared PNPP substrate (add 1 silver (Tris Buffer) and 1 gold (PNPP) tablets to 10 ml of ddH2O). Cover plate with aluminum foil, incubate 60 min in dark at RT.

  12. Read in ELISA plate reader at 405 nm. The mean corrected absorbance (compared to control mice) is calculated using the equation: Mean corrected absorbance = Mean corrected absorbance experimental/Mean corrected absorbance control, where corrected absorbance is calculated by subtraction of background absorbance from the sample absorbance.

Our assessment of humoral responses to TG showed that mice immunized with Ad-TG had significantly higher levels hTG, mTG and mTPO autoantibodies, compared to AD-LacZ-immunized mice, demonstrating the development of autoimmune thyroiditis (17).

REAGENTS AND SOLUTIONS:

10% glycerol in Phosphate buffer saline (PBS)

For 100 μl of solution, add 10 μl of glycerol (Fisher, cat. no. A16205.AP) to 90 μl Phosphate buffer saline (PBS) (ThermoFisher, cat. no. J61196.AP), mix. Keep at RT.

Ammonium-chloride-potassium buffer

For 500 ml of buffer, mix 4 g NH4Cl, 0.5 g KHCO3 and 100 μl EDTA and top up with distilled H2O up to 500 ml, adjust the pH to 7.2–7.4.

Complete RPMI 1640 medium for mouse splenocytes

For 500 ml of medium, add 50 ml (10%) Fetal bovine serum (Fisher, cat. no. A5256) and 5 ml (1%) Sodium pyruvate (Fisher, cat. no. 11360070) to 445 ml RPMI 1640 medium (Corning, cat. no. 10040CV), mix, filter.

0.1% bovine serum albumin (BSA) in PBS

For 100 ml of solution, add 0.1 g of BSA in 100 ml of PBS, mix.

Carbonate-Bicarbonate buffer

To make 100 ml of buffer, dissolve the content of 1 capsule of Carbonate-Bicarbonate powder (Fisher, cat. no. 50-100-88) in 100 ml ddH2O (final pH = 9.6).

COMMENTARY:

Background Information:

To study the etiology of Hashimoto thyroiditis (HT) different experimental approaches have been developed to induce autoimmune thyroiditis (EAT) in mice (2). The EAT mouse model was achieved by immunization of susceptible mouse strains (CBA/J, NOD) with TG, a known thyroid autoantigen, in combination with an adjuvant to break immune tolerance. This classical EAT model is widely used to study the pathoetiology of HT (2, 18). This model, similar to HT, is characterized by lymphocytic infiltration of the thyroid, follicular destruction, T cell proliferative responses against thyroglobulin (TG), and production of anti-TG autoantibodies (18). Although this is an excellent model for HT, it has limited utility in testing the functional role of TG polymorphisms that were shown to confer susceptibility to AITD (14). Therefore, to better understand the contribution of the TG AITD-susceptible variants to thyroid autoimmunity, we generated a mouse model that is easily amenable to modifications of the TG sequence. In our new model, an adenovirus vector carrying full-length hTG cDNA is used to induce B cell and T cell responses against TG in the absence of adjuvants. Moreover, the Ad-TG EAT mouse model is suitable for studying the interactions of the susceptible TG variants with environmental factors known to increase risk of AITD development, or with variants of other AITD susceptible genes (19, 20).

Critical Parameters and Troubleshooting (see Tables 2, 3)

Table 2.

Troubleshooting Guide for Immunohistochemistry assay

Problem Possible Cause Solution
High background staining Secondary antibody cross-reactivity or nonspecific binding is present Increase the serum concentration, alternatively, decrease the concentration of the secondary antibody.
Issues with the primary antibody, which may show an affinity for similar epitopes on non-target antigens Reduce the concentration of the primary antibody, adjust the concentration of triton-x in the blocking solution, or use a different primary antibody.
Weak target staining Poor primary antibody potency (due to protein degradation/denaturation caused by long-term storage, repetitive freeze/thaw cycles, microbial contamination, changes in pH, etc. Ensure that the antibody is correctly stored (at −20C or 4C) according to the manufacturer’s instructions in small aliquots, use sterile pipette tips and tubes.
Open in a new tab

Table 3.

Troubleshooting Guide for the TG ELISA assay

Problem Possible Cause Solution
Poor standard curve Inaccurate pipetting Check pipettes
Improper standard dilution Prior to opening, briefly spin the stock standard tube and dissolve the powder thoroughly by gentle mixing
Low sensitivity Improper serum dilution; problems with secondary antibody Make sure that the serum is properly stored at −20C; Increase the dilution of the serum; Increase the concentration of the secondary antibody.
Open in a new tab

Understanding Results:

In this protocol, mice were intramuscularly injected with either the adenoviral vector containing full-length hTG or control LacZ cDNA in the thigh muscle at three different time points: day 0, day 21, and day 42. The mice were euthanized on day 63, and dissection, organ harvesting, and blood collection were performed. Hematoxylin and eosin-stained thyroid sections from Ad-TG immunized mice exhibited lymphocytic infiltration, while sections from control mice showed normal thyroid structure. Immunostaining revealed a significant increase in CD4+ T-cells and CD8+ T-cells as well as B220+ B-cells in the thyroid infiltrates of Ad-TG immunized mice compared to the control AdLacZ group. These results indicate that immunization with Ad-TG triggered immune cell infiltration of the thyroid, a hallmark of experimental autoimmune thyroiditis.

Figure 5. Immunostaining of CD4+ T-cells in thyroid sections from Ad-TG and Ad-LacZ immunized NOD.H-2h4 mice.

Figure 5.

Open in a new tab

(A) Immunostaining shows the absence of the CD4+ T-cells in thyroid of control mice (Ad-LacZ). (B, C) Abundant CD4+ T-cells (green) in thyroid infiltrates of Ad-TG immunized mice (Ad-TG). Images were taken using a confocal microscope (A and B – 20x, C – 40x). Green, staining for CD4+ T-cells; Blue, DAPI staining of the nuclei.

Figure 6. Immunostaining of CD8+ T-cells in thyroid sections from Ad-TG and Ad-LacZ immunized NOD.H-2h4 mice.

Figure 6.

Open in a new tab

(A) Thyroid section from control mice (Ad-LacZ) stained with anti-CD8 Abs. (B, C) Abundant CD8+ T-cells (red) in thyroid infiltrates of Ad-TG immunized mice (Ad-TG). Images were taken using a confocal microscope (A and B – 20x, C – 40x). Red, staining for CD8+ T-cells; Blue, DAPI staining of the nuclei.

Table 4.

Time considerations for protocols (see Table 4)

Protocol Time Consideration
Basic protocol 1 63 days
Support protocol 1 1 day
Support protocol 2 5 days
Support protocol 3 2 days
Support protocol 4 2 days
Support protocol 5 3 days
Support protocol 6 2 days
Open in a new tab

ACKNOWLEDGEMENTS:

The authors thank Dr. Sandra McLachlan (Cedars-Sinai Medical Center) for generously providing the NOD.H-2h4 mice. They also thank Drs. Glaucia Furtado and Sergio Lira (Icahn School of Medicine at Mount Sinai) for sharing and advise with the immunostaining protocol. This work was supported in part by grants DK067555, DK073681, and DK129664 from NIDDK and CA280749 from NCI (to YT) and grant DK129664 from NIDDK and CA280749 from NCI (to MSL). The National Cancer Institute’s cancer center support grant P30CA013330 partially supported work that was done through the Analytical Imaging Facility and Flow Cytometry Core Facility. The Leica SP8 confocal microscope was purchased using the Shared Instrumentation Grant 1S10OD18218-1.

CONFLICT OF INTEREST STATEMENT:

Yaron Tomer was previously (1/2015–6/2017) the PI on a basic research project jointly funded by the Juvenile Diabetes Research Foundation and Pfizer. The current article is not related with that research project. The other authors have no potential conflict of interests.

Footnotes

INTERNET RESOURCES:

Figure 1 and Figure 2 were created using BioRender.com.

LITERATURE CITED:

  • 1.Tomer Y, Huber A. The etiology of autoimmune thyroid disease: a story of genes and environment. J Autoimmun. 2009;32(3–4):231–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.McLachlan SM, Rapoport B. Breaking tolerance to thyroid antigens: changing concepts in thyroid autoimmunity. Endocr Rev. 2014;35(1):59–105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Eschler DC, Hasham A, Tomer Y. Cutting edge: the etiology of autoimmune thyroid diseases. Clin Rev Allergy Immunol. 2011;41(2):190–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Chen CR, Hamidi S, Braley-Mullen H, Nagayama Y, Bresee C, Aliesky HA, et al. Antibodies to thyroid peroxidase arise spontaneously with age in NOD.H-2h4 mice and appear after thyroglobulin antibodies. Endocrinology. 2010;151(9):4583–93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Tomer Y, Ban Y, Concepcion E, Barbesino G, Villanueva R, Greenberg DA, et al. Common and unique susceptibility loci in Graves and Hashimoto diseases: results of whole-genome screening in a data set of 102 multiplex families. Am J Hum Genet. 2003;73(4):736–47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Ban Y, Tozaki T, Taniyama M, Tomita M, Ban Y. Association of a thyroglobulin gene polymorphism with Hashimoto’s thyroiditis in the Japanese population. Clin Endocrinol (Oxf). 2004;61(2):263–8. [DOI] [PubMed] [Google Scholar]
  • 7.Hsiao JY, Hsieh MC, Tien KJ, Hsu SC, Shin SJ, Lin SR. Association between a C/T polymorphism in exon 33 of the thyroglobulin gene is associated with relapse of Graves’ hyperthyroidism after antithyroid withdrawal in Taiwanese. J Clin Endocrinol Metab. 2007;92(8):3197–201. [DOI] [PubMed] [Google Scholar]
  • 8.Hsiao JY, Hsieh MC, Tien KJ, Hsu SC, Lin SR, Ke DS. Exon 33 T/T genotype of the thyroglobulin gene is a susceptibility gene for Graves’ disease in Taiwanese and exon 12 C/C genotype protects against it. Clin Exp Med. 2008;8(1):17–21. [DOI] [PubMed] [Google Scholar]
  • 9.Tomer Y, Greenberg DA, Concepcion E, Ban Y, Davies TF. Thyroglobulin is a thyroid specific gene for the familial autoimmune thyroid diseases. J Clin Endocrinol Metab. 2002;87(1):404–7. [DOI] [PubMed] [Google Scholar]
  • 10.Ban Y, Greenberg DA, Davies TF, Jacobson E, Concepcion E, Tomer Y. ‘Linkage analysis of thyroid antibody production: evidence for shared susceptibility to clinical autoimmune thyroid disease. J Clin Endocrinol Metab. 2008;93(9):3589–96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Caputo M, Rivolta CM, Mories T, Corrales JJ, Galindo P, Gonzalez-Sarmiento R, et al. Analysis of thyroglobulin gene polymorphisms in patients with autoimmune thyroiditis. Endocrine. 2010;37(3):389–95. [DOI] [PubMed] [Google Scholar]
  • 12.Mizuma T, Watanabe M, Inoue N, Arakawa Y, Tomari S, Hidaka Y, et al. Association of the polymorphisms in the gene encoding thyroglobulin with the development and prognosis of autoimmune thyroid disease. Autoimmunity. 2017;50(6):386–92. [DOI] [PubMed] [Google Scholar]
  • 13.Maierhaba M, Zhang JA, Yu ZY, Wang Y, Xiao WX, Quan Y, et al. Association of the thyroglobulin gene polymorphism with autoimmune thyroid disease in Chinese population. Endocrine. 2008;33(3):294–9. [DOI] [PubMed] [Google Scholar]
  • 14.Ban Y, Greenberg DA, Concepcion E, Skrabanek L, Villanueva R, Tomer Y. Amino acid substitutions in the thyroglobulin gene are associated with susceptibility to human and murine autoimmune thyroid disease. Proc Natl Acad Sci U S A. 2003;100(25):15119–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zhang ML, Zhang DM, Wang CE, Chen XL, Liu FZ, Yang JX. Association between thyroglobulin polymorphisms and autoimmune thyroid disease: a systematic review and meta-analysis of case-control studies. Genes Immun. 2019;20(6):484–92. [DOI] [PubMed] [Google Scholar]
  • 16.Hodge SE, Ban Y, Strug LJ, Greenberg DA, Davies TF, Concepcion ES, et al. Possible interaction between HLA-DRbeta1 and thyroglobulin variants in Graves’ disease. Thyroid. 2006;16(4):351–5. [DOI] [PubMed] [Google Scholar]
  • 17.Faustino LC, Li CW, Stefan-Lifshitz M, Kim K, Clarke OB, Tomer Y. A Novel Mouse Model of Autoimmune Thyroiditis Induced by Immunization with Adenovirus Containing Full-Length Thyroglobulin cDNA: Implications to Genetic Studies of Thyroid Autoimmunity. Thyroid. 2020;30(9):1338–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Kong YC. Experimental autoimmune thyroiditis in the mouse. Curr Protoc Immunol. 2007;Chapter 15:Unit 15 7. [DOI] [PubMed] [Google Scholar]
  • 19.Li CW, Menconi F, Osman R, Mezei M, Jacobson EM, Concepcion E, et al. Identifying a Small Molecule Blocking Antigen Presentation in Autoimmune Thyroiditis. J Biol Chem. 2016;291(8):4079–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Li CW, Osman R, Menconi F, Concepcion ES, Tomer Y. Flexible peptide recognition by HLA-DR triggers specific autoimmune T-cell responses in autoimmune thyroiditis and diabetes. J Autoimmun. 2017;76:1–9. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES