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Endocrinology, doi:10.1210/en.2004-0530
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Endocrinology Vol. 145, No. 12 5504-5514
Copyright © 2004 by The Endocrine Society

"Hijacking" the Thyrotropin Receptor: A Chimeric Receptor-Lysosome Associated Membrane Protein Enhances Deoxyribonucleic Acid Vaccination and Induces Graves’ Hyperthyroidism

Pavel N. Pichurin, Gregorio D. Chazenbalk, Holly Aliesky, Oxana Pichurina, Basil Rapoport and Sandra M. McLachlan

Autoimmune Disease Unit, Cedars-Sinai Research Institute and University of California, Los Angeles School of Medicine, Los Angeles, California 90048

Address all correspondence and requests for reprints to: Sandra M. McLachlan, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, California 90048. E-mail: mclachlans{at}cshs.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Naked DNA vaccination with the TSH receptor (TSHR) does not, in most studies, induce TSHR antibodies and never induces hyperthyroidism in BALB/c mice. Proteins expressed endogenously by vaccination are preferentially presented by major histocompatibility complex class I, but optimal T cell help for antibody production requires lysosomal processing and major histocompatibility complex class II presentation. To divert protein expression to lysosomes, we constructed a plasmid with the TSHR ectodomain spliced between the signal peptide and transmembrane-intracellular region of lysosome-associated membrane protein (LAMP)-1, a lysosome-associated membrane protein. BALB/c mice pretreated with cardiotoxin were primed intramuscularly using this LAMP-TSHR chimera and boosted twice with DNA encoding wild-type TSHR, TSHR A-subunit, or LAMP-TSHR. With each protocol, spleen cells responded to TSHR antigen by secreting interferon-{gamma}, and 60% or more mice had TSHR antibodies detectable by ELISA. TSH binding inhibitory activity was present in seven, four, and two of 10 mice boosted with TSHR A-subunit, LAMP-TSHR, or wild-type TSHR, respectively. Importantly, six of 30 mice had elevated T4 levels and goiter (5 of 6 with detectable thyroid-stimulating antibodies). Injecting LAMP-TSHR intradermally without cardiotoxin pretreatment induced TSHR antibodies detectable by ELISA but not by TSH binding inhibitory activity, and none became hyperthyroid. These findings are consistent with a role for cardiotoxin-recruited macrophages in which (unlike in fibroblasts) LAMP-TSHR can be expressed intracellularly and on the cell surface. In conclusion, hijacking the TSHR to lysosomes enhances T cell responses and TSHR antibody generation and induces Graves’-like hyperthyroidism in BALB/c mice by intramuscular naked DNA vaccination.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OUTCOME OF naked DNA vaccination encoding the human TSH receptor (TSHR) is variable. Graves’-like hyperthyroidism was induced in 17% of outbred mice (1) and approximately 30% of nonobese diabetic (NOD) mice transgenic for the major histocompatibility complex (MHC) class II allele HLA-DR3 (2). However, on a mixed C57BL/10 background, HLA-DR3 transgenic mice developed TSHR antibodies but not hyperthyroidism (3). BALB/c mice have been intensively investigated because of their well-known propensity to generate antibodies. In one study (4), virtually all TSHR-DNA vaccinated mice of this strain developed TSHR antibodies. In contrast, we and others (5, 6, 7, 8) reported undetectable or low levels of TSHR antibodies in the majority of genetically immunized BALB/c mice. However, even in BALB/c mice with TSHR antibodies induced by conventional DNA vaccination, few had thyroid-stimulating antibodies (TSAbs) and none were hyperthyroid (4).

Variations in response to TSHR-DNA vaccination reflect different genetic backgrounds and possibly also different vaccination protocols, such as cardiotoxin pretreatment vs. DNA in sucrose, single vs. multiple injections, and housing animals in conventional vs. pathogen-free facilities. Because of the potential for vaccine development, immunologists have explored ways of enhancing immune responses to genetic immunization. One modification involves injecting DNA for the antigen together with DNA-encoding cytokines (for example, see Ref.9). However, covaccination with TSHR-DNA and DNA-encoding granulocyte-macrophage colony-stimulating factor or IL-4 did not increase the incidence of TSHR antibodies or hyperthyroidism (2).

A more fundamental problem concerns the presentation to T cells of peptides from an antigen transiently expressed by DNA vaccination. Endogenous proteins (like intracellular viral proteins) are degraded to linear peptides in the proteasome and transported by transporters in antigen processing molecules for binding to MHC class I molecules (reviewed in Ref.10). Infected target cells that present viral peptides in MHC class I are recognized and killed by cytotoxic CD8+ T cells. In contrast, exogenous proteins (like bacterial products) are internalized by phagocytosis or pinocytosis and transported to lysosomes in which they are processed into peptides and bind to MHC class II (reviewed in Ref.11). MHC class I vs. MHC class II processing and presentation is not mutually exclusive. However, reduced MHC class II presentation of endogenously processed peptides translates into diminished CD4+ T cell activation, reduced help for B cells, and less antibody secretion. Against this background, it is not surprising that DNA vaccination induces cytotoxic T cells but is less powerful for inducing antibodies. Indeed, we could readily demonstrate memory T cell responses in antibody-negative mice vaccinated using TSHR-DNA (5, 6, 12).

An alternative strategy to cytokine immune enhancement involves targeting the antigen or autoantigen to a particular intracellular compartment. The lysosome-associated membrane protein (LAMP)-1 has a sorting signal that directs it to lysosomes (13). Consequently, this molecule has been used as a tool to direct proteins to the lysosome that would not normally enter this pathway. For example, the acetylcholine receptor (AChR){alpha} subunit cDNA has been substituted for that of LAMP-1 between the signal peptide and transmembrane/cytoplasmic tail of the latter. Antigen-presenting cells transfected with this chimeric AChR-LAMP-1 DNA were more potent T cell stimulators than the same cells transfected with the AChR{alpha} subunit alone (14). In the present study, we constructed a chimeric LAMP-TSHR plasmid and tested its efficacy for intramuscular DNA vaccination. In addition, we explored the outcome of intradermal DNA vaccination with the LAMP-TSHR plasmid because of the reported efficacy of this route for inducing antibodies (15). Remarkably, by hijacking the TSHR to the lysosomes, we induced functional TSHR antibodies and hyperthyroidism in BALB/c mice by DNA vaccination.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of LAMP-TSHR ectodomain chimera
A plasmid containing the Torpedo AChR{alpha} subunit spliced between the LAMP1 signal peptide and transmembrane/cytoplasmic domains (pcDNA3.1D-SigTACHRLAMP1) (14) was kindly provided by Dr. Daniel Drachman (Department of Neurology, Johns Hopkins University, Baltimore, MD). With SigTACHRLAMP1 and a plasmid containing the TSHR cDNA (16) as templates, we used overlapping PCR to substitute the TSHR cDNA ectodomain (amino acid residues 22–417) for the AChR subunit, retaining the LAMP1 signal peptide and transmembrane/cytoplasmic domains. For this purpose we generated the following DNA fragments using Pfu DNA polymerase (Stratagene, La Jolla, CA): 1) a 150-bp fragment encoding the LAMP1 signal peptide and the first 11 amino acids of the TSHR (residues 22–33, amino acids 1–21 being the TSHR signal peptide); a KpnI restriction site was included in the upstream oligonucleotide; and 2) a 1.2-kb fragment encoding the C terminal 11 residues of the LAMP-signal peptide, the TSHR ectodomain (amino acids 22–417), and a 3' EcoRI restriction site incorporated in the downstream oligonucleotide. The two fragments were annealed and overlapping PCR performed to generate 5' KpnI-LAMP1 signal peptide-TSHR ectodomain-3' EcoRI. After restriction with KpnI and EcoRI, this combined fragment was ligated into the same sites in pcDNA3.1D-SigTACHRLAMP1 (for simplicity renamed LAMP-TSHR) to replace the AChR{alpha} subunit. Nucleotide sequencing confirmed the insertion sites and amplification fidelity of LAMP-TSHR.

Intracellular expression of LAMP-TSHR was tested in transiently transfected Chinese hamster ovary (CHO) K1 cells by flow cytometry. To provide positive and negative controls, CHO-K1 cells were transfected with the wild-type TSHR in pcDNA3.1 (5) and pcDNA3.1D-SigTACHRLAMP1, respectively. The protocol for intracellular TSHR expression was described previously (17). Briefly, 24 h after transfection, adherent cells were lifted from their plastic supports (Ca/Mg-free PBS followed by trypsin), fixed in chilled 1% paraformaldehyde and permeabilized using saponin buffer [0.01 M PBS (pH 7.4), 0.5% BSA, and 0.1% saponin, the latter from Sigma Aldrich, St. Louis, MO]. Cell aliquots were incubated with mouse monoclonal TSHR antibody 2C11 (Serotec Ltd., Oxford UK) or second antibody only, and binding was detected using fluorescein isothiocyanate-conjugated antimouse IgG (Caltag Laboratories Inc., Burlingame, CA). Flow cytometry was performed using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA).

Immunization by DNA vaccination
Intramuscular (im) DNA vaccination was performed as previously described (5). In brief, female BALB/c mice (Jackson Laboratories, Bar Harbor, ME) aged 6–7 wk were pretreated in the quadriceps muscle with cardiotoxin (100 µl/injection,10 µM Naja nigricollis, Calbiochem, La Jolla, CA). Five to seven days later, the mice were injected in the same muscle with one of three plasmids (100 µg DNA in 50 µl): LAMP-TSHR DNA (see above); DNA encoding the full-length wild-type TSHR (WT-TSHR) in pcDNA3.1 (6); or TSHR amino acid residues 1–289, essentially the TSHR A-subunit (18). The vaccination protocol was performed three times at intervals of 3 wk. Where indicated, some mice were vaccinated intradermally (id) without cardiotoxin pretreatment by injecting LAMP-TSHR DNA (25, 50, and 100 µg in 50 µl) at four to five sites at the base of the tail. Four weeks after the third vaccination (im or id), mice were euthanized to obtain blood, thyroid glands, and spleens. All animal studies were approved by the Institutional Animal Care and Use Committee and performed with the highest standards of care in a pathogen-free facility.

TSHR antigen and TSHR peptides
TSHR A-subunit protein (TSHR-289) (18) secreted into culture medium by CHO cells was purified by affinity chromatography using mouse monoclonal antibody 3BD10, as previously described (19). Purity and concentration were analyzed by SDS-PAGE. Before use in ELISA or lymphocyte cultures (below), TSHR A-subunit protein was dialyzed against 10 mM Tris (pH 7.4), 50 mM NaCl.

The panel of 26 peptides spanning the extracellular domain of the TSHR was previously described (20). Peptides were synthesized using an automated 431A peptide synthesizer and HPLC purified. Each peptide is 20 amino acids long and overlaps the next peptide by five amino acids. Peptides corresponding to amino acids 22–41, 37–56, 52–71, etc. are designated A, B, C, etc. (residues 1–21 include the signal peptide that is cleaved from the mature TSHR protein). Two extracellular loop peptides (residues 471–494 and 661–571) are designated EC1 and EC2. Peptides were resuspended in sterile distilled water and used at a final concentration of 10 µg/ml.

Splenocyte responses to TSHR antigen and peptides
Splenocytes (duplicate 200-µl aliquots of ~5 x 105 cells) were incubated in round-bottomed 96-well plates in the presence or absence of TSHR-antigen (TSHR A-subunit, see above; 10 µg/ml), TSHR peptides (10 µg/ml), or Concanavalin A (5 µg/ml; Sigma Aldrich). Culture medium was RPMI 1640, 10% heat inactivated fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 50 µg/ml gentamicin, 50 µM ß-mercaptoethanol, and 100 U/ml penicillin. After 5–6 d (37 C, 5% CO2), culture supernatants were centrifuged to remove cell debris and stored at –80 C. Supernatants were assayed for interferon (IFN){gamma} by ELISA (100 µl; in duplicate) using capture and biotinylated detection antibodies (BD PharMingen, San Diego, CA).

ELISA for TSHR antibodies
ELISA wells coated with TSHR A-subunit protein [1 µg/ml in 10 mM Tris (pH 7.4), 50 mM NaCl] were incubated with test sera (duplicate aliquots, diluted 1:100 and 1:1000) as previously described (5). Antibody binding was detected with horseradish peroxidase-conjugated mouse anti-IgG (Sigma Aldrich), the signal developed with o-phenylenediamine and H2O2, and OD read at 490 nm.

Inhibition of 125I-TSH binding to the TSHR (TBI)
The ability of TSHR antibodies to inhibit TSH binding to the TSHR (TBI) (21) was measured using a kit (Kronus, Boise, ID). Serum aliquots (25 µl; undiluted) were incubated with detergent solubilized porcine TSHR; 125I-TSH (bovine) was added, and the TSHR-antibody complexes were precipitated with polyethylene glycol. The TBI values are calculated from the following formula:

TSAbs
TSAb was measured as previously described (22). In brief, monolayers of CHO cells expressing the wild-type TSHR in 96-well plates were incubated with 3% test serum in 100 µl Hank’s buffer without NaCl and supplemented with 20 mM HEPES (pH 7.4), 1.5 mM isobutylmethylxanthine, 220 mM sucrose, 4% polyethylene glycol 4000, and 0.3% BSA (all from Sigma Aldrich). After 2 h at 37 C, total cAMP content (medium and cells) was measured by RIA. TSAb was expressed as a percentage of basal cAMP generated in the presence of serum from a control DNA immunized mouse.

Serum T4 and thyroid histology
Total T4 in mouse sera was measured in undiluted serum (25 µl) by RIA using a kit (Diagnostic Products Corp., Los Angeles, CA). Thyroid glands were fixed in 4% paraformaldehyde (Sigma Aldrich) (pH 7.5). Serial sections were prepared from paraffin sections, stained with hematoxylin and eosin, and examined for lymphocytic infiltrate.

Statistical analysis
Differences between responses of mice were determined by rank sum tests or ANOVA on ranks. Sera from individual mice were considered positive in the TBI or TSAb assays or to have elevated T4 levels, whether the values exceeded the mean + 2 SD of the values for control-DNA vaccinated mice.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of LAMP-TSHR
Expression of the TSHR-LAMP chimera was tested by transiently transfecting CHO cells with DNA for this construct. As measured by intracellular flow cytometry using monoclonal antibody 2C11, TSHR expression was detected in CHO cells transfected with DNA for LAMP-TSHR and wild-type TSHR but not in CHO cells transfected with the LAMP1-AChR plasmid (Fig. 1Go, arrows).



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  		FIG. 1. Flow cytometry demonstrating intracellular expression of the LAMP-TSHR chimera. This chimeric molecule contains the TSHR ectodomain sandwiched between the LAMP-1 signal peptide and the LAMP-1 transmembrane/cytoplasmic domain. CHO-K1 cells were transiently transfected with plasmids encoding the LAMP-TSHR chimera (A), wild-type TSHR (B), or AChR-LAMP chimera (C) (14 ). After fixation and permeabilization, cells were stained with TSHR-specific antibody (2C11) or second antibody only. Antibody binding was detected by flow cytometry using fluorescein isothiocyanate-conjugated antimouse IgG. Arrows indicate intracellular expression of the TSHR (A and B but not C).

 
Experimental design
We used three DNA plasmids to vaccinate BALB/c mice (Fig. 2AGo): 1) DNA encoding the LAMP-TSHR, which should direct expression of the TSHR ectodomain to the MHC class II pathway; 2) DNA coding for the full-length TSHR (6); and 3) DNA encoding the TSHR A-subunit (18).



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  		FIG. 2. TSHR plasmids and protocols for vaccinating BALB/c mice. A, Three cDNAs in the vector pcDNA3.1 were used: the LAMP-TSHR; TSHR with its own signal peptide, transmembrane, and cytoplasmic region; and the TSHR A-subunit (A-sub) with the TSHR-signal peptide and TSHR ectodomain amino acid (aa) residues 22–289 (18 ). B, Vaccination protocols. For im vaccination, mice were pretreated with cardiotoxin and about 5 d later injected as follows: one group was primed with LAMP-TSHR and boosted with two injections of wild-type (WT) TSHR DNA; the second group was primed with LAMP-TSHR DNA and injected twice with TSHR A-subunit DNA; the third group received three injections of LAMP-TSHR. In earlier studies, mice were injected im three times with TSHR DNA (5 6 ). For id injection, mice were injected three times with LAMP-TSHR without cardiotoxin pretreatment. The interval between vaccinations was 3 wk.

 
The first step for inducing antibody responses is activating naive CD4+ T cells (see introductory section). Therefore, BALB/c mice (pretreated with cardiotoxin) were primed with one im injection of LAMP-TSHR DNA (Fig. 2BGo) and divided into three groups. One group of mice was boosted with two im vaccinations of DNA encoding the full-length TSHR. The second group received two injections of TSHR A-subunit DNA. The third group received two further injections of the priming plasmid (the LAMP-TSHR chimera), a protocol that we hypothesized would induce cellular immunity but not TSHR antibodies, at least to the native TSHR. The outcome of these three vaccination protocols was compared with our previous approach, namely vaccinating BALB/c mice three times with the wild-type TSHR (5, 6).

In addition, we determined the effect of id vaccination using LAMP-TSHR DNA. As in other studies using the id approach, the animals were vaccinated three times without cardiotoxin pretreatment (Fig. 2BGo). When the mice were euthanized, they were tested for splenocyte memory responses to TSHR antigen, TSHR antibodies, and thyroid function.

Responses to TSHR antigen and TSHR peptides by splenocytes from mice vaccinated im
To assess cellular immunity, splenocytes were cultured with TSHR A-subunit antigen, and responses were assessed by measuring IFN{gamma} production. Splenocytes from all groups of mice primed with LAMP-TSHR DNA produced significantly higher levels of IFN{gamma} than control DNA-injected mice (Fig. 3AGo, P < 0.05, ANOVA on ranks).



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  		FIG. 3. Splenocyte responses to challenge with TSHR antigen and TSHR peptides in mice vaccinated im. A, Responses to TSHR A-subunit protein (10 µg/ml) by splenocytes from individual mice assessed by IFN{gamma} production (picomoles per milliliter/ml). Three groups of mice were primed with LAMP-TSHR DNA (LAMP-TSHR). One group was boosted twice with the same LAMP-TSHR plasmid (n = 6 mice). A second group was boosted twice with TSHR-WT DNA (TSHR, n = 10) and a third group with TSHR A-subunit DNA (A-sub, n = 9). Control (Con) mice (n = 5) received three injections of empty vector DNA. *, Values significantly different (P < 0.05, ANOVA on ranks). B, Responses to TSHR peptides. Peptides A to Z encompass the TSHR ectodomain and peptides EC1 and EC2 correspond to extracellular loops 1 and 2 (see Methods). Five mice primed with LAMP-TSHR DNA were studied, two animals boosted twice with LAMP-TSHR and three mice boosted twice with wild-type TSHR DNA. Splenocytes from each mouse were studied individually. IFN{gamma} production by splenocytes was expressed as a percentage of the maximum peptide response in each mouse. These percentage data were then combined and used to plot the bar graph (mean + SEM; n = 5). *, Responses to peptides C and J significantly greater than responses in medium (M) alone (P < 0.05, ANOVA on ranks). C, IFN{gamma} production (picomoles per milliliter) by splenocytes from the five mice studied in B. Mean values (+ SEM) are shown for lymphocytes cultured in medium (M) and in the presence of TSHR A-subunit protein (A-sub). Included is the mean + SEM for the maximum peptide responses (Pep) used to calculate the percentages in B.

 
Next, we studied the TSHR peptides recognized by splenocytes from mice vaccinated three times with LAMP-TSHR DNA and mice primed with LAMP-TSHR and vaccinated twice with wild-type TSHR DNA. Because of the variability in splenocyte responses between individual mice (see Fig. 3AGo), responses were expressed as a percentage of the maximum amount of IFN{gamma} generated by a peptide in each mouse. These percentages were pooled (n = 5 mice) and used to plot a bar graph (Fig. 3BGo). Of the 28 TSHR peptides, C and J induced splenocyte responses in all five mice (ANOVA on ranks, P < 0.05). Previously we found the same peptides to be immunodominant in BALB/c mice vaccinated with the wild-type TSHR-DNA, with no response to these or other peptides by splenocytes from control mice (12). To provide insight into the magnitude of peptide responses, IFN{gamma} generated by the peptide inducing the maximum response (mean + SEM) was compared with splenocyte IFN{gamma} production to TSHR A-subunit antigen (Fig. 3CGo). Overall, priming with LAMP-TSHR DNA induced strong memory T cell responses to TSHR antigen with no change in TSHR peptide recognition.

Intramuscular DNA vaccination with LAMP-TSHR, unlike wild-type TSHR, induces TSHR antibodies
We confirmed our hypothesis that priming with a plasmid-directing TSHR expression to the lysosome would facilitate a TSHR antibody response. After priming, most mice boosted with DNA for the wild-type TSHR or the TSHR A-subunit were antibody positive by ELISA (1:100; Fig. 4AGo). Surprisingly, however, most mice both primed and boosted with LAMP-TSHR DNA also developed TSHR antibodies. Moreover, the titer of these TSHR antibodies in some mice was sufficient for detection even at a dilution of 1:1000 (Fig. 4BGo). Included in Fig. 4AGo are our previous data for 38 mice vaccinated with the unmodified, wild-type TSHR plasmid. TSHR antibodies were undetectable by ELISA in the majority of these animals, with only two mice being antibody positive (6).



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  		FIG. 4. Vaccinating BALB/c mice im with LAMP-TSHR DNA induces TSHR antibodies detectable by ELISA. After priming with LAMP-TSHR, mice were boosted twice with DNA encoding wild-type TSHR (n = 10); TSHR A-subunit (A-sub, n = 10); or LAMP-TSHR (n = 10). Antibodies were measured in sera diluted 1:100 (A) and 1:1000 (B). Also included are the data for control-DNA-vaccinated mice (n = 5) and previously reported values for 38 BALB/c mice vaccinated with TSHR-DNA, only two of which had antibodies clearly positive by ELISA (5 6 ). *, Values significantly greater than for control-DNA-vaccinated mice (P < 0.05, ANOVA on ranks).

 
LAMP-TSHR DNA priming im induces functional TSHR antibodies and hyperthyroidism
Next, we measured antibodies with TBI activity and TSAb activity in mice primed with LAMP-TSHR and boosted using three different TSHR-DNAs. The majority of mice (seven of 10) boosted with TSHR A-subunit DNA had elevated TBI levels, compared with control DNA-vaccinated mice (Fig. 5AGo). Despite high TSHR antibody levels in ELISA (Fig. 4Go), TBI values were significantly lower in mice boosted with the wild-type TSHR, compared with mice boosted with TSHR A-subunit, with only two of 10 being marginally TBI positive (Fig. 5AGo; P < 0.038, rank sum test). An intermediate number (four of 10) of mice vaccinated three times with LAMP-TSHR were positive for TBI.



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  		FIG. 5. TBI, TSAb, T4, and thyroid histology in BALB/c mice vaccinated im using LAMP-TSHR. Mice were primed with LAMP-TSHR DNA and boosted twice with DNA encoding wild-type TSHR (n = 10); TSHR A-subunit (A-sub) (n = 10); and LAMP-TSHR (n = 10). The shaded areas in A, B, and C correspond to the mean ± 2 SD of values for sera from control-DNA-vaccinated mice (n = 4, A and B; n = 7, C). A, TBI activity expressed as percent inhibition of maximal TSH binding in the presence of control serum. *, Values significantly greater in mice boosted with TSHR A-subunit than mice boosted with wild-type TSHR (P < 0.038, rank sum test). B, TSAb activity expressed as percent cAMP values for control serum. *, Values significantly greater in mice boosted with DNA for TSHR A-subunit or LAMP-TSHR vs. mice boosted wild-type TSHR DNA (P < 0.05, ANOVA on ranks). C, Total T4 levels (micrograms per deciliter). Of the mice primed with TSHR-LAMP, five animals had serum T4 elevated above mean + 2 SD for controls: namely, three mice, two mice, and one mouse from the groups boosted with TSHR A-subunit, TSHR-LAMP, and wild-type TSHR, respectively. D and E, Representative thyroid epithelial morphology. D, Mouse vaccinated with control DNA. E, Mouse with hyperthyroidism induced by priming with LAMP-TSHR and boosting twice with the same plasmid. Unlike the gland in the euthyroid animal, thyrocytes in the TSHR-immunized mouse are more columnar and vacuolated, consistent with hyperthyroidism. Magnification, x100.

 
TSAb activity followed the general pattern for TBI activity. Compared with control DNA-vaccinated mice, TSAb was detectable in six of 10 mice boosted with TSHR A-subunit, none of 10 boosted with wild-type TSHR DNA, and three of 10 mice boosted with LAMP-TSHR DNA (Fig. 5BGo). Of the nine TSAb-positive animals, five mice had TSAb activity exceeding 250% cAMP generated by control serum, of which three animals had been boosted with TSHR A-subunit DNA and one had been boosted with LAMP-TSHR DNA.

Turning to thyroid function, serum total T4 levels were elevated, compared with controls in three of 10 mice boosted with TSHR A-subunit DNA and two of 10 mice vaccinated three times with LAMP-TSHR DNA (Fig. 5CGo). These five hyperthyroid mice had the highest TSAb levels (Fig. 5BGo). In addition, one of 10 mice boosted with TSHR wild-type DNA was marginally hyperthyroid (Fig. 5CGo). When the mice were euthanized, goiters were present in mice later found to have elevated levels of TSAb and T4. Microscopically, compared with euthyroid controls, these hyperthyroid glands had cuboidal thyrocytes with marked intracellular vacuolation, indicating higher secretory activity (Fig. 5Go, D and E). Overall, 25% (five of 20) of mice primed with LAMP-TSHR and boosted with either TSHR A-subunit or LAMP-TSHR developed many characteristics of Graves’ disease, namely goiter, TSAb activity, elevated T4, and follicular hyperactivity with hyperplasia.

Intradermal vaccination with LAMP-TSHR DNA
The above experiments established the efficacy of im LAMP-TSHR DNA vaccination for inducing TSHR antibodies. As mentioned previously, id vaccination is reported to be more effective than im injection for generating antibody responses (15). We, therefore, investigated whether vaccination via this route would be more powerful than im vaccination with LAMP-TSHR DNA. Moreover, because id delivery might require less DNA than the standard im dose (100 µg), we tested three different doses, namely 25, 50, and 100 µg. As in other studies using the id approach, the animals were vaccinated without cardiotoxin pretreatment on three occasions (Fig. 2BGo).

Mice vaccinated id with LAMP-TSHR DNA developed T cell responses to TSHR A-subunit protein (Fig. 6AGo), comparable with those in mice immunized im with the same plasmid (Fig. 3AGo). Moreover, virtually all animals had TSHR antibodies detectable by ELISA in sera diluted 1:100 and some also at 1:1000 (Fig. 6BGo). The mean ELISA values in mice vaccinated id using 25 µg of plasmid were similar to those in mice vaccinated im with 100 µg LAMP-TSHR, namely 1.89 ± 0.67 vs. 1.86 ± 0.50, respectively (OD 490 nm; mean ± SEM). Although the mean TSHR antibody OD values were somewhat higher in mice injected id with 50 and 100 µg (2.73 ± 0.75 and 2.53 ± 0.57, respectively), none of the differences were statistically significant.



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  		FIG. 6. BALB/c mice vaccinated id with LAMP-TSHR DNA develop splenocyte responses to TSHR antigen and TSHR antibodies detectable by ELISA. Mice were injected three times with increasing doses of LAMP-TSHR (25, 50, or 100 µg; five mice per group) without cardiotoxin pretreatment. Control mice (Con) were vaccinated with empty vector DNA (A, four mice; B, five mice). A, Responses to TSHR A-subunit protein (10 µg/ml) by splenocytes from individual mice assessed by IFN{gamma} production (picomoles per milliliter). *, Values significantly greater than for control DNA-vaccinated mice (P < 0.03, rank sum test). B, TSHR antibodies measured by ELISA in sera diluted 1:100 and 1:1000.

 
Despite high TSHR antibody levels by ELISA, there was little evidence for functional TSHR antibody activity in id vaccinated mice. TBI activity was detectable in three of five mice from the 25-µg group, one of five mice in the 50-µg group, and none of five mice vaccinated with 100 µg (Fig. 7AGo). Moreover, the highest of these TBI values was much lower than the maximum level in mice vaccinated im with the same plasmid (~25% vs. > 90%; Fig. 7AGo vs. Fig. 5AGo). Despite the presence of some antibodies capable of inhibiting TSH binding, only one mouse vaccinated id with LAMP-TSHR was positive for TSAb (Fig. 7BGo). None of these 15 mice had visible goiters when euthanized, nor did they have elevated serum T4 levels (Fig. 7CGo).



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  		FIG. 7. TBI, TSAb, and T4 in BALB/c mice vaccinated id with LAMP-TSHR DNA. Mice were injected three times with LAMP-TSHR (25, 50, or 100 µg; five mice in each group) without cardiotoxin pretreatment. Control mice (Con) were vaccinated with empty vector DNA (n = 4, A and B; n = 7 C). A, TBI activity expressed as percent inhibition of TSH binding. B, TSAb activity expressed as percent cAMP values for control serum. C, T4 levels (microgram per deciliter) measured in undiluted sera. In all panels, the shaded areas correspond to the mean ± 2 SD for sera from control DNA-vaccinated mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The initial promise of naked TSHR-DNA vaccination in BALB/c mice for generating antibodies to the native TSHR (4) was not fulfilled when this approach was tested by other groups (5, 6, 7, 8). Indeed, although a monoclonal antibody with TSAb activity was recently isolated from a BALB/c mouse vaccinated with TSHR-DNA (23), hyperthyroidism did not develop in this strain by this immunization protocol (4). A potential problem with DNA vaccination for antibody induction is that TSHR expressed by DNA is processed and presented predominantly as an endogenous protein by MHC class I, a pathway likely to stimulate the wrong T cell subset, probably CD8+ cells. To circumvent this problem, we constructed a plasmid (LAMP-TSHR) (Fig. 2AGo) using components of the lysosome-associated membrane protein to direct TSHR expression to lysosomes and thereby bias TSHR peptide presentation to MHC class II. We hypothesized that priming with LAMP-TSHR would stimulate CD4+ T cells that, on boosting with DNA for the wild-type TSHR (or the TSHR-A subunit), would provide help to B cells encountering native TSHR protein. Indeed, this approach generated higher TSHR antibody levels in significantly more mice than we previously reported using TSHR DNA alone (5, 6). Moreover, by hijacking the TSHR to MHC class II pathway, we show that im DNA vaccination induces TSAb, hyperthyroidism, and goiter in approximately 20% of BALB/c mice.

Some of our findings are consistent with previous evidence for LAMP’s ability to substantially enhance immune responses. As mentioned previously, antigen presenting cells transfected with chimeric LAMP-AChR{alpha} subunit were more potent stimulators of T cells in vitro than the same cells transfected with the AChR{alpha} subunit alone (14). In other examples examining in vivo responses, immunity after DNA vaccination was enhanced using a Dengue virus construct with the transmembrane and cytoplasmic regions of envelope genes replaced by those of LAMP (24). Likewise, a DNA vaccine for human papilloma virus (HPV-16 E7) employing LAMP-1 enhanced antitumor effects against an E7-expressing tumor (25). Even using vaccinia virus (far more immunogenic than naked DNA), greater E7-specific immune responses were induced using LAMP-associated HPV-16 E7 gene than with the wild-type HPV-16 E7 (26).

In terms of the epitopes recognized by T cells, differences have been reported between peptides processed from endogenous or exogenous hen egg lysozyme (27, 28). However, when ovalbumin was targeted to the lysosome using the LAMP-1 transmembrane/cytoplasmic domain, presentation of only one peptide was increased, compared with exogenous ovalbumin assessed using recognition by T cell hybridomas (29). Our findings are consistent with the ovalbumin data. Thus, splenocytes primed with LAMP-TSHR and boosted with wild-type TSHR or LAMP-TSHR responded to the same peptides (C and J) as BALB/c mice vaccinated three times with wild-type TSHR DNA (12).

Overall, after im priming with LAMP-TSHR, our observation of enhanced antibody production on boosting with plasmids expressing the wild-type TSHR or the TSHR A-subunit was consistent with prediction. However, one finding was unexpected. We anticipated that immunization with LAMP-TSHR alone would enhance T cell responses but not induce TSHR antibodies. The reason for this expectation is that a LAMP-TSHR protein should be degraded in the lysosome, providing peptides for T cell recognition but no intact TSHR protein to drive B cell antibody production. The fact that TSHR antibodies were generated suggests that TSHR antigen released from damaged cells in vivo could provide a target for B cell activation and the production of antibodies to denatured antigen. However, even more unexpectedly, some antibodies had TBI activity and even TSAb associated with hyperthyroidism. These findings raise the important question of the nature of TSHR antigen required in vivo to induce thyroid stimulating antibodies.

Conformationally intact TSHR antigen is required for human TSHR autoantibody recognition (reviewed in Ref.30). Multiple attempts by us and others to generate antigenically suitable recombinant TSHR indicated that intracellularly retained TSHR lack the conformation required for recognition by functional TSHR antibodies (reviewed in Ref.30). LAMP-1 is rapidly transported directly to lysosomes, although early studies demonstrated that a small proportion reaches the cell surface (31). More recently it has been shown that activated macrophages, but not fibroblasts, express posttranslationally glycosylated LAMP-1 on their surface (32). At this location, LAMP-1 acts as a costimulatory molecule, termed M150. The LAMP-TSHR chimera lacks the M150 protein and cannot play a role in costimulation. However, antigen capable of inducing functional TSHR antibodies could be provided in the form of glycosylated TSHR ectodomain expressed on the macrophage cell surface by LAMP-TSHR DNA.

When injected id, the LAMP-TSHR chimera induced T cell responses and TSHR antibodies detected by ELISA, consistent with the well-established efficacy of id DNA vaccination for antibody generation. However, these TSHR antibodies had low or undetectable TBI activity; only one mouse was TSAb positive, and no mice vaccinated in this manner were hyperthyroid. Unlike im vaccination, id injection was performed without cardiotoxin pretreatment. Muscle damage by the toxin likely attracts macrophages and/or dendritic cells to the site at which DNA is subsequently injected. Moreover, following institutional guidelines, we performed id injections at the base of the tail, a region with a paucity of dendritic cells (for example, see Ref.33). Very recently in a study published during revision of our manuscript (34), TSAb and hyperthyroidism were induced in approximately 20% of BALB/c mice vaccinated id with TSHR DNA. Of note, the DNA was injected into the skin on the back, not at the tail base, of the mice. Taking this information into account, we suggest a possible explanation for our observation that id LAMP-TSHR does not induce hyperthyroidism: in the absence of dendritic cells, most LAMP-TSHR protein is intracellular and lacks the conformation necessary to induce biologically active TSHR antibodies.

Another point of interest is the difference in functional antibodies and hyperthyroidism between mice boosted with wild-type TSHR vs. TSHR A-subunit DNA. When expressed by an adenovirus vector, the TSHR-A subunit induced significantly more hyperthyroidism than the wild-type TSHR, despite comparable TSHR antibody levels measured by ELISA and TBI (22). In the present study, we found that, despite similar TSHR antibody levels by ELISA, mice boosted with TSHR A-subunit DNA had significantly higher TBI activity than mice boosted with wild-type TSHR DNA. It should be appreciated that the LAMP-TSHR chimera contains the TSHR ectodomain and lacks the seven membrane-spanning segments of the wild-type TSHR. Previously we demonstrated that Graves’ autoantibodies preferentially recognize the TSHR ectodomain tethered by a glycosylphosphatidylinositol anchor, compared with the same ectodomain tethered to the serpentine region of the wild-type TSHR (35). We suggest that, like the TSHR-ectodomain-glycosylphosphatidylinositol, the TSHR ectodomain expressed by LAMP-TSHR is more accessible to drive TSAb-specific B cells.

Despite the advantages of LAMP-TSHR over the wild-type TSHR for DNA vaccination, this approach is less effective than injecting the TSHR (or its A-subunit) im in an adenovirus vector (7, 22). Several factors are likely to account for the difference. First, 125I TSH binding was detectable in muscle membranes from mice injected with TSHR adenovirus but not with TSHR-DNA plasmid (7). These findings reflect stronger TSHR expression using adenovirus than vaccination with naked DNA. Second, macrophages/dendritic cells (rather than muscle cells) must be transfected for cell surface expression of LAMP-TSHR encoded by plasmid. Adenovirus itself induces tissue damage, and macrophages and dendritic cells are recruited to the injection site. However, transfecting macrophages/dendritic cells requires injecting cardiotoxin some days earlier to recruit immune cells to the site subsequently used to inject LAMP-TSHR DNA. Technical variability in injecting twice into the same area, combined with lower TSHR expression, may explain in part the lesser ability of LAMP-TSHR DNA, compared with TSHR-adenovirus, to induce hyperthyroidism. A final factor not to be overlooked is the more potent adjuvant effect of adenovirus (36), compared with DNA CpG motifs (37), which may compensate for reduced MHC class II presentation of endogenously processed peptides.

In conclusion, we have shown that hijacking the TSHR to the MHC class II pathway using a LAMP-TSHR chimeric DNA increases memory T cell responses and greatly enhances TSHR antibody generation. Using this LAMP-TSHR plasmid, together with cardiotoxin pretreatment, we demonstrate the induction of Graves’-like disease in BALB/c mice by im naked DNA vaccination.


   Acknowledgments
 
We thank Dr. Daniel Drachman (Department of Neurology, Johns Hopkins School of Medicine, Baltimore, MD) for providing us with the plasmid pcDNA3.1D-SigTACHRLAMP1, Dr. Alexei Miagkov (Department of Neurology, Johns Hopkins School of Medicine) for advice on constructing LAMP-TSHR, Dr. John Morris (Mayo Clinic, Rochester, MN) for providing the TSHR synthetic peptides, and Dr. Boris Catz (Los Angeles, CA) for his generous support.


   Footnotes
 
This work was supported by National Institutes of Health Grants DK 54684 (to S.M.M.), DK 19289 (to B.R.), and a Winnick Family Clinical Research Scholar Award (to S.M.M.).

Abbreviations: AChR, Acetylcholine receptor; CHO, Chinese hamster ovary; HPV, human papilloma virus; id, intradermal; IFN, interferon; LAMP, lysosome-associated membrane protein; MHC, major histocompatibility complex; TBI, inhibition of TSH binding to the TSHR; TSAb, thyroid-stimulating antibody; TSHR, TSH receptor.

Received April 26, 2004.

Accepted for publication August 16, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Costagliola S, Many MC, Denef JF, Pohlenz J, Refetoff S, Vassart G 2000 Genetic immunization of outbred mice with thyrotropin receptor cDNA provides a model of Graves’ disease. J Clin Invest 105:803–811[Medline]
  2. Flynn JC, Rao PV, Gora M, Alsharabi G, Wei W, Giraldo AA, David CS, Banga JP, Kong YM 2004 Graves’ hyperthyroidism and thyroiditis in HLA-DRB1*0301 (DR3) transgenic mice after immunization with thyrotropin receptor DNA. Clin Exp Immunol 135:35–40[CrossRef][Medline]
  3. Pichurin P, Chen CR, Pichurina O, David C, Rapoport B, McLachlan SM 2003 Thyrotropin receptor-DNA vaccination of transgenic mice expressing HLA-DR3 or HLA-DQ6b. Thyroid 13:911–917[CrossRef][Medline]
  4. Costagliola S, Rodien P, Many M-C, Ludgate M, Vassart G 1998 Genetic immunization against the human thyrotropin receptor causes thyroiditis and allows production of monoclonal antibodies recognizing the native receptor. J Immunol 160:1458–1465[Abstract/Free Full Text]
  5. Pichurin P, Yan X-M, Farilla L, Guo J, Chazenbalk G, Rapoport B, McLachlan SM 2001 Naked thyrotropin receptor DNA vaccination: A TH1 T cell response in which interferon-{gamma} production, rather than antibody, dominates the immune response in mice. Endocrinology 142:3530–3536[Abstract/Free Full Text]
  6. Pichurin P, Pichurina O, Chazenbalk GD, Paras C, Chen CR, Rapoport B, McLachlan SM 2002 Immune deviation away from Th1 in interferon-{gamma} knockout mice does not enhance TSH receptor antibody production after naked DNA vaccination. Endocrinology 143:1182–1189[Abstract/Free Full Text]
  7. Nagayama Y, Kita-Furuyama M, Ando T, Nakao K, Mizuguchi H, Hayakawa T, Eguchi K, Niwa M 2002 A novel murine model of Graves’ hyperthyroidism with intramuscular injection of adenovirus expressing the thyrotropin receptor. J Immunol 168:2789–2794[Abstract/Free Full Text]
  8. Rao PV, Watson PF, Weetman AP, Carayanniotis G, Banga JP 2003 Contrasting activities of thyrotropin receptor antibodies in experimental models of Graves’ disease induced by injection of transfected fibroblasts or deoxyribonucleic acid vaccination. Endocrinology 144:260–266[Abstract/Free Full Text]
  9. Pan CH, Chen HW, Tao MH 1999 Modulation of immune responses to DNA vaccines by codelivery of cytokine genes. J Formos Med Assoc 98:722–729[Medline]
  10. Heemels MT, Ploegh H 1995 Generation, translocation, and presentation of MHC class I-restricted peptides. Annu Rev Biochem 64:463–491[CrossRef][Medline]
  11. Bona C 2004 Processing and presentation of self antigens. In: Theofilopoulos AN, Bona CA, eds. The molecular pathology of autoimmune diseases. New York: Taylor and Francis; 184–191
  12. Pichurin P, Schwarz-Lauer L, Braley-Mullen H, Paras C, Pichurina O, Morris JC, Rapoport B, McLachlan SM 2002 Peptide scanning for thyrotropin receptor T-cell epitopes in mice vaccinated with naked DNA. Thyroid 12:755–764[CrossRef][Medline]
  13. Guarnieri FG, Arterburn LM, Penno MB, Cha Y, August JT 1993 The motif Tyr-X-X-hydrophobic residue mediates lysosomal membrane targeting of lysosome-associated membrane protein 1. J Biol Chem 268:1941–1946[Abstract/Free Full Text]
  14. Wu JM, Wu B, Guarnieri F, August JT, Drachman DB 2000 Targeting antigen-specific T cells by genetically engineered antigen presenting cells. A strategy for specific immunotherapy of autoimmune disease. J Neuroimmunol 106:145–153[CrossRef][Medline]
  15. Boyle JS, Silva A, Brady JL, Lew AM 1997 DNA immunization: induction of higher avidity antibody and effect of route on T cell cytotoxicity. Proc Natl Acad Sci USA 94:14626–14631[Abstract/Free Full Text]
  16. Nagayama Y, Kaufman KD, Seto P, Rapoport B 1989 Molecular cloning, sequence and functional expression of the cDNA for the human thyrotropin receptor. Biochem Biophys Res Commun 165:1184–1190[CrossRef][Medline]
  17. Chen C-R, Tanaka K, Chazenbalk GD, McLachlan SM, Rapoport B 2001 A full biological response to autoantibodies in Graves’ disease requires a disulfide-bond loop in the thyrotropin N-terminus homologous to a laminin EGF-like domain. J Biol Chem 276:14767–14772[Abstract/Free Full Text]
  18. Chazenbalk GD, Jaume JC, McLachlan SM, Rapoport B 1997 Engineering the human thyrotropin receptor ectodomain from a nonsecreted form to a secreted, highly immunoreactive glycoprotein that neutralizes autoantibodies in Graves’ patients’ sera. J Biol Chem 272:18959–18965[Abstract/Free Full Text]
  19. Chazenbalk G, McLachlan S, Pichurin P, Rapoport B 2001 A "prion-like" shift between two conformational forms of a recombinant thyrotropin receptor A subunit module: purification and stabilization using chemical chaperones of the form reactive with Graves’ autoantibodies. J Clin Endocrinol Metab 86:1287–1293[Abstract/Free Full Text]
  20. Morris JC, Bergert ER, McCormick DJ 1993 Structure-function studies of the human thyrotropin receptor. Inhibition of binding of labeled thyrotropin (TSH) by synthetic human TSH receptor peptides. J Biol Chem 268:10900–10905[Abstract/Free Full Text]
  21. Shewring GA, Rees Smith B 1982 An improved radioreceptor assay for TSH receptor antibodies. Clin Endocrinol (Oxf) 17:409–417[Medline]
  22. Chen C-R, Pichurin P, Nagayama Y, Latrofa F, Rapoport B, McLachlan SM 2003 The thyrotropin receptor autoantigen in Graves’ disease is the culprit as well as the victim. J Clin Invest 111:1897–1904[CrossRef][Medline]
  23. Costagliola S, Franssen JD, Bonomi M, Urizar E, Willnich M, Bergmann A, Vassart G 2002 Generation of a mouse monoclonal TSH receptor antibody with stimulating activity. Biochem Biophys Res Commun 299:891–896[CrossRef][Medline]
  24. Raviprakash K, Marques E, Ewing D, Lu Y, Phillips I, Porter KR, Kochel TJ, August TJ, Hayes CG, Murphy GS 2001 Synergistic neutralizing antibody response to a dengue virus type 2 DNA vaccine by incorporation of lysosome-associated membrane protein sequences and use of plasmid expressing GM-CSF. Virology 290:74–82[CrossRef][Medline]
  25. Kim JW, Hung CF, Juang J, He L, Kim TW, Armstrong DK, Pai SI, Chen PJ, Lin CT, Boyd DA, Wu TC 2004 Comparison of HPV DNA vaccines employing intracellular targeting strategies. Gene Ther 11:1011–1118[CrossRef][Medline]
  26. Chen CH, Wang TL, Hung CF, Pardoll DM, Wu TC 2000 Boosting with recombinant vaccinia increases HPV-16 E7-specific T cell precursor frequencies of HPV-16 E7-expressing DNA vaccines. Vaccine 18:2015–2022[CrossRef][Medline]
  27. Moreno J, Vignali DA, Nadimi F, Fuchs S, Adorini L, Hammerling GJ 1991 Processing of an endogenous protein can generate MHC class II-restricted T cell determinants distinct from those derived from exogenous antigen. J Immunol 147:3306–3313[Abstract]
  28. Bonifaz LC, Arzate S, Moreno J 1999 Endogenous and exogenous forms of the same antigen are processed from different pools to bind MHC class II molecules in endocytic compartments. Eur J Immunol 29:119–131[CrossRef][Medline]
  29. Fernandes DM, Vidard L, Rock KL 2000 Characterization of MHC class II-presented peptides generated from an antigen targeted to different endocytic compartments. Eur J Immunol 30:2333–2343[CrossRef][Medline]
  30. Rapoport B, Chazenbalk GD, Jaume JC, McLachlan SM 1998 The thyrotropin receptor: interaction with thyrotropin and autoantibodies. Endocr Rev 19:673–716[Abstract/Free Full Text]
  31. Carlsson SR, Fukuda M 1992 The lysosomal membrane glycoprotein lamp-1 is transported to lysosomes by two alternative pathways. Arch Biochem Biophys 296:630–639[CrossRef][Medline]
  32. Prasad DV, Parekh VV, Joshi BN, Banerjee PP, Parab PB, Chattopadhyay S, Kumar A, Mishra GC 2002 The Th1-specific costimulatory molecule, m150, is a posttranslational isoform of lysosome-associated membrane protein-1. J Immunol 169:1801–1809[Abstract/Free Full Text]
  33. Bergstresser PR, Fletcher CR, Streilein JW 1980 Surface densities of Langerhans cells in relation to rodent epidermal sites with special immunologic properties. J Invest Dermatol 74:77–80[CrossRef][Medline]
  34. Barrett K, Liakata E, Rao PV, Watson PF, Weetman AP, Lymberi P, Banga JP, Carayanniotis G 2004 Induction of hyperthyroidism in mice by intradermal immunization with DNA encoding the thyrotropin receptor. Clin Exp Immunol 136:413–422[CrossRef][Medline]
  35. Chazenbalk GD, Pichurin P, Chen CR, Latrofa F, Johnstone AP, McLachlan SM, Rapoport B 2002 Thyroid-stimulating autoantibodies in Graves disease preferentially recognize the free A subunit, not the thyrotropin holoreceptor. J Clin Invest 110:209–217[CrossRef][Medline]
  36. Geutskens SB, van der Eb MM, Plomp AC, Jonges LE, Cramer SJ, Ensink NG, Kuppen PJ, Hoeben RC 2000 Recombinant adenoviral vectors have adjuvant activity and stimulate T cell responses against tumor cells. Gene Ther 7:1410–1416[CrossRef][Medline]
  37. Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, Koretzky GA, Klinman DM 1995 CpG motifs in bacterial DNA trigger direct B cell activation. Nature 374:546–549[CrossRef][Medline]



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S. M. McLachlan, Y. Nagayama, and B. Rapoport
Insight into Graves' Hyperthyroidism from Animal Models
Endocr. Rev., October 1, 2005; 26(6): 800 - 832.
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