Critical Differences between Induced and Spontaneous Mouse Models of Graves' Disease with Implications for Antigen-Specific Immunotherapy in Humans

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1 This information is current as of April 9, Subscription Permissions Alerts Why The JI? Submit online. Critical Differences between Induced and Spontaneous Mouse Models of Graves' Disease with Implications for Antigen-Specific Immunotherapy in Humans Basil Rapoport, Bianca Banuelos, Holly A. Aliesky, Nicole Hartwig Trier and Sandra M. McLachlan J Immunol published online 9 November ol Rapid Reviews! 30 days* from submission to initial decision No Triage! Every submission reviewed by practicing scientists Fast Publication! 4 weeks from acceptance to publication *average Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: Downloaded from by guest on April 9, 2018 The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD Copyright 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: Online ISSN:

2 Published November 9, 2016, doi: /jimmunol The Journal of Immunology Critical Differences between Induced and Spontaneous Mouse Models of Graves Disease with Implications for Antigen-Specific Immunotherapy in Humans Basil Rapoport,* Bianca Banuelos,* Holly A. Aliesky,* Nicole Hartwig Trier, and Sandra M. McLachlan* Graves hyperthyroidism, a common autoimmune disease caused by pathogenic autoantibodies to the thyrotropin (TSH) receptor (TSHR), can be treated but not cured. This single autoantigenic target makes Graves disease a prime candidate for Ag-specific immunotherapy. Previously, in an induced mouse model, injecting TSHR A-subunit protein attenuated hyperthyroidism by diverting pathogenic TSHR Abs to a nonfunctional variety. In this study, we explored the possibility of a similar diversion in a mouse model that spontaneously develops pathogenic TSHR autoantibodies, NOD.H2 h4 mice with the human (h) TSHR (htshr) A-subunit transgene expressed in the thyroid and (shown in this article) the thymus. We hypothesized that such diversion would occur after injection of inactive htshr A-subunit protein recognized only by nonpathogenic (not pathogenic) TSHR Abs. Surprisingly, rather than attenuating the pre-existing pathogenic TSHR level, in TSHR/NOD.H2 h4 mice inactive htshr Ag injected without adjuvant enhanced the levels of pathogenic TSH-binding inhibition and thyroid-stimulating Abs, as well as nonpathogenic Abs detected by ELISA. This effect was TSHR specific because spontaneously occurring autoantibodies to thyroglobulin and thyroid peroxidase were unaffected. As controls, nontransgenic NOD.H2 h4 mice similarly injected with inactive htshr A-subunit protein unexpectedly developed TSHR Abs, but only of the nonpathogenic variety detected by ELISA. Our observations highlight critical differences between induced and spontaneous mouse models of Graves disease with implications for potential immunotherapy in humans. In htshr/nod.h2 h4 mice with ongoing disease, injecting inactive htshr A-subunit protein fails to divert the autoantibody response to a nonpathogenic form. Indeed, such therapy is likely to enhance pathogenic Ab production and exacerbate Graves disease in humans. The Journal of Immunology, 2016, 197: G raves hyperthyroidism is directly caused by pathogenic autoantibodies to the thyrotropin (TSH) receptor (TSHR) that mimic the stimulatory effects of TSH (reviewed in Ref. 1). The disease can be treated, but there is no cure for the underlying autoimmune process. Thyroid ablation with radioiodine, the most common therapy in the United States (2), results, almost inevitably, in permanent hypothyroidism requiring lifelong thyroid hormone ingestion. Thiourea drugs are effective at inhibiting thyroid hormone synthesis (3) and can be used for years, but in the majority of cases the disease recurs when the drugs are discontinued. Several novel therapeutic approaches for human Graves disease are being tested including small-molecule inhibitors of TSHR function (4, 5), monoclonal TSHR Abs that block the function of *Thyroid Autoimmune Disease Unit, Cedars-Sinai Medical Center and UCLA School of Medicine, Los Angeles, CA 90048; and Department of Autoimmunology and Biomarkers, Statens Serum Institut, DK-2300 Copenhagen S, Denmark ORCID: (N.H.T.). Received for publication August 10, Accepted for publication October 14, This work was supported by National Institutes of Health Grants DK (to S.M.M.) and DK19289 (to B.R.). Address correspondence and reprint requests to Dr. Sandra M. McLachlan, Thyroid Autoimmune Disease Unit, Cedars-Sinai Medical Center, 8400 Beverly Boulevard B-131, Los Angeles, CA address: mclachlans@cshs.org Abbreviations used in this article: CHO, Chinese hamster ovary; h, human; htshr, human TSHR; PEG, polyethylene glycol 4000; TBI, TSH-binding inhibition; Tg, thyroglobulin; TgAb, thyroglobulin Ab; TPO, thyroid peroxidase; TPOAb, thyroid peroxidase Ab; TSAb, thyroid-stimulating Ab; TSH, thyrotropin; TSHR, thyrotropin receptor. Copyright Ó 2016 by The American Association of Immunologists, Inc /16/$30.00 thyroid-stimulating autoantibodies (6), and inhibitors of components of the adaptive immune system, such as rituximab, which target B lymphocytes (7, 8). Most important, however, even if successfully introduced into the pharmacopeia, these approaches may treat but will not cure Graves disease, and nonspecific immunological inhibitors have potentially severe adverse effects. Ag-specific immunotherapy has long been attempted for autoimmune conditions such as multiple sclerosis and type 1 diabetes mellitus, but clinical trials have been disappointing. An editorial (9) on the limited therapeutic efficacy of myelin basic protein peptide immunotherapy for multiple sclerosis suggested that the limited efficacy was ascribed to the wide spectrum of Ags targeted by the immune system in this disease and others like type 1 diabetes mellitus (10), leading to the suggestion that in attempts to cure autoimmune diseases, it may be advantageous to focus on rarer diseases in which the immune response in largely limited to a single Ag. There is no need to search for a rare disease. Graves disease is one of the most common autoimmune diseases affecting humans withaprevalencerateof 1% (11). Moreover, it is the prime example of an autoimmune disease directly caused by autoimmunity to a single autoantigen, the TSHR. In mice with induced hyperthyroidism, a number of novel therapeutic approaches have been attempted, including a shift from Th1 to Th2 CD4 + T cells (or vice versa) induced by various agents (12 14), blockade of TNF family ligand inhibitors (BAFF and APRIL) (15), anti-cd20 mab (rituximab) (16), immunoproteasome inhibition (17), small-molecule antagonism of the TSHR (4), and injection of purified, recombinant TSHR protein (18). Of these approaches, only purified, recombinant TSHR protein was both Ag-specific and targeted the immune system with the goal

3 2 ATTEMPTED Ag-SPECIFIC THERAPY IN MURINE GRAVES DISEASE of inducing tolerance to the TSHR (18). However, success with this approach was limited. In the induced Graves disease model using adenovirus expressing the TSHR A-subunit, prior injection of A-subunit protein attenuated the development of hyperthyroidism but was ineffective in reversing hyperthyroidism once established (18). Disease attenuation occurred with eukaryotic, not prokaryotic, A-subunit protein but, contrary to expectation, it was not associated with reduced TSHR tolerance. Instead, there was a diversion from bioactive to nonfunctional TSHR Ab. Ab diversion has been used to treat experimentally induced myasthenia gravis in rats by injecting pathologically irrelevant epitopes on the cytoplasmic domains of the acetylcholine receptor (19). Graves disease develops spontaneously in humans. Therefore, in this study, we focused on a mouse model that spontaneously develops pathogenic TSHR Abs, namely NOD.H2 h4 mice with the human (h) TSHR (htshr) A-subunit transgene targeted to the thyroid (TSHR/NOD.H2 h4 ) (20). In this study, htshr A-subunit transgenic mice and their nontransgenic NOD.H2 h4 littermates were injected two or three times with htshr A-subunit protein in the absence of adjuvant. Our findings were most unexpected and emphasize the critical importance of using a spontaneous model, rather than an induced model, in attempting to apply autoantigenspecific treatment to deviate pathogenic TSHR Abs. Materials and Methods Mouse strains and injection of TSHR A-subunit protein or OVA Transgenic NOD.H2 h4 mice expressing the htshr A-subunit (TSHR/ NOD.H2 h4 ) (20) and nontransgenic NOD.H2 h4 littermates were bred at Cedars-Sinai Medical Center. Mice of the TSHR/NOD.H2 h4 strain have been cryopreserved by the Mutant Mouse Regional Resource Center under the designation NOD.Cg_Tg(TG_TSHR)51.9Smcl. Wild type BALB/cJ mice were purchased from Jackson Laboratories (Bar Harbor, ME). The novel transgenic strain was derived by crossing BALB/c mice expressing low levels of the htshr A-subunit in the thyroid and thymus (21 23) with NOD.H2 h4 mice, repeated backcrossing of the transgenic progeny to wild-type NOD.H2 h4 for eight generations (N8) (20). The TSHR/NOD.H2 h4 mice used in this study were from the N10 and N11 generations (.99.9% NOD.H2 h4 genome). To emphasize that the transgene encodes the htshr A-subunit, we will refer to this strain as htshr/ NOD.H2 h4 mice. htshr/nod.h2 h4 (n = 37) and nontransgenic NOD.H2 h4 littermates (n = 30) were injected s.c. on the back with htshr A-subunit protein (prepared as described later) or saline. At the indicated internals, mice aged 8 wk received three injections of htshr A-subunit protein (4, 10, and 10 mg), and in another series, mice aged 12 wk received two injections of 10 mg of htshr A-subunit protein (Fig. 1). It must be emphasized that the htshr A-subunit protein was injected without adjuvant. As controls, BALB/c mice aged 8 wk (five males, five females) were injected three times at the indicated intervals with htshr A-subunit protein (4, 10, and 10 mg) without adjuvant. Additional BALB/c mice were studied without ha-subunit protein injections. As a further control, a separate group of nontransgenic NOD.H2 h4 mice (six males, five females) was injected s.c. on the back with OVA (Sigma Chemical, St. Louis, MO) following the same time intervals and doses as for A-subunit protein. From the age of 9 wk, all mice (htshr/nod.h2 h4, nontransgenic NOD.H2 h4, and BALB/c) were provided with sodium iodide supplemented drinking water (0.05% NaI). Blood was drawn at 17 and 25 wk, and mice were euthanized after 41 wk (Fig. 1). These time intervals correspond to 8-, 16-, and 32-wk exposure to NaI. Mouse studies were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Cedars-Sinai Medical Center and were carried out with the highest standards of care in a pathogen-free facility. TSHR A-subunit protein Recombinant htshr A-subunit protein (aa residues ) secreted by Chinese hamster ovary (CHO) cells with an amplified transgenome (24) was purified from culture supernatants by affinity chromatography (25) and dialyzed against Tris saline (10 mm Tris, ph 7.4, 50 mm NaCl). Two different conformational forms of this recombinant htshr protein can be purified separately from the conditioned culture medium based on their reciprocal recognition by Graves patients autoantibodies and mouse mab 3BD10: active htshr A-subunits only by the former and inactive htshr A-subunits only by the latter (26). To preclude the possibility of passive TSHR Ab neutralization, we used inactive htshr-289, hereafter referred to as ha-subunit protein. TSHR Ab assays TSHR Abs were measured using three assays. ELISA. The assay for ELISATSHR Abs (IgG class) was reported previously (27). ELISA wells were coated with ha-subunit protein (inactive form, described earlier; 5 mg/ml) and incubated with test sera (1:100 dilution). The positive controls were serum from BALB/c mice immunized with htshr A-subunit adenovirus (e.g., Ref. 28) and the mab 3BD10 (25). Ab binding was detected with HRP-conjugated mouse anti-igg (A 3673; Sigma Chemical), and the signal was developed with o-phenylenediamine and H 2 O 2. Data are reported as the OD at 490 nm. TSH-binding inhibition assay. TSH-binding inhibition (TBI) levels were measured in 25 ml of mouse serum using a clinical assay kit (Kronus, Star, ID). The data are reported as the percentage inhibition of [ 125 I]-TSH binding to the TSH holoreceptor. Bioassay for thyroid-stimulating Ab. A bioassay was used to measure camp generation by CHO cells expressing the htshr (27, 29). To permit testing 10 ml of mouse serum per well, we used the following approach: 25 ml of test mouse serum + 75 ml of normal human serum ( carrier protein ) was precipitated with 300 ml of 20% polyethylene glycol 4000 (PEG; Sigma- Aldrich) in water. Because of the large amount of carrier protein needed for PEG precipitation, we used normal human serum (one batch from a single donor in all assays) as described previously (e.g., Ref. 29). The negative controls were sera from BALB/c mice; for the positive control, sera from normal BALB/c mice were supplemented with human monoclonal TSHR M22 (30). Negative and positive control samples were precipitated using PEG in the same way using normal human serum as carrier protein. The pellets were resuspended in 240 ml of Ham s F12 medium supplemented with 10 mm of Hepes (ph 7.4), 1 mm of isobutylmethylxanthine, and 0.3% BSA. Duplicate aliquots (110 ml) were applied to htshr-expressing CHO cell monolayers in 96-well plates. After incubation (90 min, 37 C), the medium was aspirated, intracellular camp was extracted with ethanol, evaporated to dryness, and resuspended in 0.2 ml of Dulbecco s PBS. Aliquots (12 ml) were assayed using the LANCE camp kit (PerkinElmer, Boston, MA). Thyroid-stimulating Ab (TSAb) activity was expressed as a percentage of camp values attained with PEG precipitated IgG from BALB/c mice. Autoantibodies to thyroglobulin and thyroid peroxidase Thyroglobulin (Tg) was isolated from murine thyroid glands as described previously (31). ELISA wells (Immulon 4HBX; Thermo Scientific, Rochester, NY) were coated with mouse Tg (1.5 mg/ml) and incubated with test sera (duplicate aliquots, 1:100 dilution). Ab binding was detected with HRP-conjugated goat anti-mouse IgG (A3673; Sigma Chemical), the signal developed with o-phenylenediamine, and the reaction stopped using 20% H 2 SO 4. The negative control was serum from 8-wk-old NOD.H2 h4 mice on regular water; the positive control was serum from BALB/c mice immunized with mouse Tg and CFA (28). Thyroglobulin Ab (TgAb) data are presented as the OD at 490 nm. Thyroid peroxidase (TPO) Abs (TPOAbs) were measured by flow cytometry using CHO cells stably expressing mouse TPO as previously described (31). A cell-based assay was used rather than ELISA because NOD.H2 h4 mice do not recognize human TPO (28) and mouse TPO generated in eukaryotic cells is not available. Sera (diluted 1:50) were incubated with mouse TPO-CHO cells; binding was detected with fluorescein isothiocyanate-conjugated affinity-purified goat anti-mouse IgG (A16073; Life Technologies, Carlsbad, CA). Cells staining with propidium iodide (1 mg/ml) were excluded from analysis. The negative control for IgG class Ab binding to mouse TPO-CHO cells was serum from 8-wk-old NOD.H2 h4 mice. Positive controls were mouse mabs 15 and 64 to human TPO (32), kindly provided to us by Dr. J. Ruf (Marseille, France), that also recognize mouse TPO (31, 32). Flow cytometry was performed (10,000 events) using a FACS Canto II with CELLQUEST Software (Becton Dickinson, San Jose, CA). Data are reported as the geometric mean. Ab response to OVA Abs to OVA were measured by ELISA as previously described (33). In brief, ELISA plates were coated with OVA (1 mg/ml) in 20 mm of Tris-HCL (ph 7.5). Duplicate serum aliquots (diluted 1:100) were applied to the wells, and Ab binding was detected with HRP-conjugated goat anti-mouse IgG (A3673; Sigma Chemical). The signal was developed with o-phenylenediamine and

4 The Journal of Immunology 3 the reaction stopped using 20% H 2 SO 4. The positive control was mouse mab HYB obtained from CF1 3 BALB/c mice immunized intraperitoneally with native OVA (33). The cutoff point for positivity was established from the binding (mean + 2 SD) to OVA-coated ELISA plates of sera from NOD.H2 h4 mice on NaI for 16 wk without OVA injections. Intrathymic expression of the endogenous mouse TSHR and the transgenic htshr A-subunit Thymuses (32-d-old mice) from htshr/nod.h2 h4 mice (n = 3) were stored in RNAlater (Life Technologies). Quantitative RT-PCR was performed essentially as previously described (23, 34). Tissue was homogenized with QIAshredder columns (Qiagen, Valencia, CA). Total RNA was prepared using RNeasy Plus Mini kit (Qiagen). The mrna samples were treated with TURBO DNase (Life Technologies) to remove genomic DNA. Reverse transcription was performed with AffinityScript QPCR cdna Synthesis Kit (Agilent Technologies, Cedar Creek, TX) using oligo(dt) and random primers. Quantitative real-time PCR was performed using the FastStart SYBR Green Master mix (Roche, Basel, Switzerland) with 2.5% of cdna (20 ml final volume). Reactions were run on an icycler Thermal Cycler with iq5 Real Time PCR Detection System module (Bio- Rad Laboratories, Hercules, CA). An initial denaturation step at 95 C (10 min) was followed by denaturation at 95 C (30 s), and annealing and extension at 55 C (30 s) for 40 cycles. Relative gene expression levels were calculated using the comparative method (DDCt), according to the Pfaffl model (35), using Bio-Rad iq5 2.0 software. Samples were tested in triplicate; parallel controls lacked reverse transcriptase. Data were normalized to mouse b-actin. For the mouse TSHR and mouse b-actin, we used mouse TSHR RT2 qprimer Assay for Mouse TSHR and RT2 qprimer Assay for Mouse Actb (Qiagen). Primers for the htshr A-subunit were as follows: sense, 59-GCAAGAAACACCTGGACTCTTAA-39; htshr A-subunit antisense, 59-GGTGGTGATGGCTAGTCTGA-39. Primers for the mouse TSHR were designed to avoid overlap with A-subunit of htshr in transgenic animals. Statistics Significant differences between responses in different groups were determined by Mann Whitney rank sum test or, when normally distributed, by Student t test. Multiple comparisons were made using ANOVA. Tests were performed using SigmaStat (Jandel Scientific Software, San Rafael, CA). Statistical results and the specific tests performed are provided in the appropriate figure legends. Results Injecting htshr A-subunit protein induces nonpathogenic TSHR Abs NOD.H2 h4 mice and enhances generation of these Abs in NOD.H2 h4 with the htshr A-subunit transgene Before attempting to attenuate the spontaneous development of pathogenic TSHR Abs, we examined the effect of htshr A-subunit protein injections (Fig. 1) on nonpathogenic TSHR Abs, which are measured by ELISA. As a control, these Abs were not induced in nontransgenic NOD.H2 h4 mice injected with saline (Fig. 2A). Unexpectedly in the absence of adjuvant, TSHR ha-subunit protein injected s.c. did induce TSHR ELISA Abs in these nontransgenic mice, detectable at 16 and 32 wk (Fig. 2B). In NOD.H2 h4 mice with the htshr A-subunit transgene (htshr/nod.h2 h4 ), TSHR ELISA Abs were detected at low levels in a few saline-injected animals at 16 and 32 wk (Fig. 2C), consistent with our former observations (20). Relative to saline controls, TSHR ELISA Ab levels were significantly FIGURE 1. Protocol for injecting htshr A-subunit protein in the absence of adjuvant. higher (at 16, but not 32 wk) in mice injected with TSHR ha-subunit protein, again without adjuvant (Fig. 2D). Similar observations were made for mice that received three injections of A-subunit protein (from 8 wk of age) or two injections (from 12 wk of age). No significant differences were observed between males and females (data not shown). These changes were specific for nonpathogenic TSHR ELISA Abs. Consistent with the known characteristics of NOD.H2 h4 mice, TgAb, first detectable at 8 wk, continued to be positive at 16 and 32 wk in virtually all animals whether nontransgenic or htshr transgenic, and regardless of whether injected with TSHR A-subunit (Fig. 3). Similarly, TPOAbs (measured after 16 and 32 wk) remained detectable in most mice and were not altered by A-subunit protein injection (Fig. 4). Intrathymic expression of the htshr A-subunit transgene and the mouse TSHR The level of intrathymic expression of the TSHR has been shown to be a susceptibility factor in human Graves disease (36, 37). It should be noted that the htshr/nod.h2 h4 mouse is not a global transgenic; the transgene was targeted to the thyroid using the tissue-specific Tg promoter. We therefore studied intrathymic expression of both the htshr A-subunit and the endogenous mouse TSHR. As determined by quantitative RT-PCR, both nontransgenic NOD.H2 h4 and transgenic htshr/nod.h2 h4 mice expressed the endogenous mouse TSHR in the thymus, but only intrathymic mrna from htshr/nod.h2 h4 transgenic mice was positive for the htshr A-subunit (Fig. 5). These data are consistent with our previous observations in low expressor TSHR A-subunit BALB/c mice (23) from which the transgene was transferred to the NOD.H2 h4 background. Responses to htshr A-subunit protein injected without adjuvant occur in BALB/c mice, and autoimmune-prone NOD.H2 h4 mice do not exhibit overreactivity The surprising finding of nonpathogenic TSHR A-subunit Ab induction in NOD.H2 h4 mice injected with ha-subunit protein without adjuvant (Fig. 2) raised the possibility that this autoimmuneprone mouse strain was unduly susceptible to loss of tolerance to the TSHR compared with other mouse strains or to the TSHR specifically. We therefore performed two additional series of ha-subunit injections without adjuvant according to the protocol outlined in Fig. 1 in non-autoimmune-prone BALB/c mice, and with an irrelevantag,ova,innod.h2 h4 mice. All BALB/c mice (males and females) developed surprisingly high levels of TSHR ELISA Abs at 16 and 32 wk after injection of htshr A-subunit protein in the absence of adjuvant (Fig. 6A). After 16 wk, mean TSHR ELISA Ab ELISA OD values were in BALB/c compared with in wild-type NOD.H2 h4 and in TSHR/NOD.H2 h4 transgenic mice. The significantly lower values in htshr/nod.h2 h4 transgenic mice than BALB/c and wild-type type littermates (p, 0.05, ANOVA) reflects greater tolerance in the transgenics, consistent with the intrathymic expression of the htshr A-subunit in this strain (Fig. 5). Incidentally, a few TSHR A-subunit protein injected BALB/c mice developed low levels of TgAb (Fig. 6B, left). In contrast, TgAb did not develop in BALB/c mice also exposed to NaI but without TSHR A-subunit protein injections (Fig. 6B, right). Turning to the outcome of OVA injection without adjuvant, OVA Abs were induced at low levels in a few NOD.H2 h4 mice (Fig. 6C). In assays standardized using mab specific for each protein, induced TSHR ELISA Ab levels were much higher in nontransgenic NOD.H2 h4 mice than OVA Ab levels induced in the same strain (Fig. 6D).

5 4 ATTEMPTED Ag-SPECIFIC THERAPY IN MURINE GRAVES DISEASE FIGURE 2. TSHR Abs measured by binding to htshr A-subunit coated ELISA plates after 8, 16, and 32 wk in nontransgenic NOD.H2 h4 mice (A and B) and htshr transgenic NOD.H2 h4 mice (C and D). Data are shown as OD 290 nm in ELISA for individual mice (males and females) injected three times with saline [(A) and (C), open circles] or with htshr A-subunit protein [(B) and (D); two injections represented by gray circles, three injections represented by solid circles]. All injections of htshr A-subunit protein were performed without adjuvant. The numbers of mice in each group ranged between 13 and 23 animals in each group. Dotted line represents mean + 2SD in nontransgenic NOD.H2 h4 mice. Values significantly different from saline-injected mice for the same time interval: *p = 0.010, **p = 0.002, ^p = (rank sum tests). Pathogenic TSHR Abs are enhanced by htshr A-subunit protein injection (without adjuvant) only in htshr/nod.h2 h4 transgenic mice The pathogenicity of TSHR Abs was first tested using in the clinical TBI assay. This study was limited to female mice because males develop very high levels of TSH that cannot be distinguished from TBI (20). Whether injected or not injected with hasubunit protein, sera from all nontransgenic NOD.H2 h4 mice were TBI-negative, except for one mouse that was borderline positive at 16 wk (Fig. 7A). In contrast, some sera from saline-injected transgenic TSH/NOD.H2 h4 mice were TBI-positive (two of six mice at 16 wk; five of seven mice at 32 wk). Moreover, only in the transgenic mice, TSHR ha-subunit protein injections enhanced TBI levels (Fig. 7B) and also increased the number of TBI-positive mice (6 of 10 at 16 wk and 9/11 at 32 wk). Notably, despite the high levels of nonpathogenic TSHR Abs detected by ELISA that developed in BALB/c mice injected with TSHR ha-subunit protein (Fig. 6A), none developed TBI activity ( % inhibition of TSH binding, mean 6 SE). FIGURE 3. TgAbs after 8, 16, and 32 wk in (A) nontransgenic NOD.H2 h4 mice and (B) htshr transgenic NOD.H2 h4 mice. Data are shown as OD 290 nm in ELISA for individual mice (both males and females). Saline injections are shown as open circles; htshr A-subunit protein injected twice (gray circles) or three times (black circles) without adjuvant. The cutoff level for TgAb positivity is shown as a dashed line (mean + 2 SD in BALB/c mice).

6 The Journal of Immunology 5 FIGURE 4. TPOAbs after 16 and 32 wk in (A) nontransgenic NOD.H2 h4 mice and (B) htshr transgenic NOD.H2 h4 mice. Males and females were studied. Data are shown for individual mice as geometric means. Saline injections are shown as white circles; TSHR A-subunit protein injected twice as gray circles or three times as black circles. Injections of htshr A-subunit protein were performed without adjuvant. The cutoff level for TPOAb positivity is shown as a dashed line (mean + 2 SD in BALB/c mice). In a second approach, sera from female mice were tested for TSHR Abs in a TSAb bioassay, the gold standard for pathogenicity. TSAb was positive in a single nontransgenic NOD.H2 h4 mouse injected with ha-subunit protein (Fig. 8A). In contrast, TSAb activity was enhanced in 5 of 11 htshr/nod.h2 h4 mice injected with TSHR ha-subunit protein in the absence of adjuvant (Fig. 8B). It should be emphasized that the ha-subunit protein used for injection was in the inactive state, that is, the form that is unable to neutralize pathogenic TSHR Abs detected by the inhibition of TSH binding to its receptor or TSAb activity. Discussion The availability of a mouse model (htshr/nod.h2 h4 mice) that spontaneously develops pathogenic TSHR autoantibodies with properties identical to those observed in human Graves disease provides a unique opportunity to investigate immunotherapeutic approaches to attenuate or reverse the generation of these Abs. Previous studies in the induced TSHR A-subunit adenovirus model showed that injecting htshr A-subunit protein without adjuvant before adenovirus immunization attenuated hyperthyroidism by deviating TSHR Abs from functional to inactive Abs (18). Turning to the spontaneous htshr/nod.h2 h4 model, in addition to pathogenic TSHR autoantibodies measured using clinical assays (TBI and TSAb), these mice also develop nonpathogenic TSHR Abs detectable by ELISA. The htshr Ag on ELISA plates is of the inactive form, not recognized by pathogenic autoantibodies and only by nonpathogenic Abs. In this study, we used these htshr/nod.h2 h4 mice to test the hypothesis that injecting inactive htshr A-subunit protein would enhance generation of nonpathogenic TSHR Abs detected by ELISA, and thereby deviate the spontaneous development of pathogenic TSHR Abs to these innocuous Abs. The data obtained did not fulfill our hypothesis, but, in contrast, provided unexpected information that will be important in future studies in both animals and humans. Surprisingly, injecting inactive ha-subunits into htshr/nod.h2 h4 transgenic mice boosted, rather than attenuated, the generation of pathogenic TSHR Abs. These changes were specific for the TSHR because the levels of Abs to autologous Tg and TPO were unchanged after injection of htshr A-subunit protein. A second observation with heuristic relevance in our study relates to the nontransgenic NOD.H2 h4 parental strain. Inactive htshr A-subunit protein injected without adjuvant led to the generation of high levels of nonpathogenic (but not pathogenic) Abs detected by ELISA. However, BALB/c mice, which are not autoimmune disease prone, behave in a similar manner to NOD.H2 h4 mice in developing high levels of nonpathogenic TSHR Abs when challenged with heterologous htshr A-subunit protein in the absence of adjuvant. Moreover, these findings were unexpected because previous studies FIGURE 5. Intrathymic expression of the transgenic htshr A-subunit and the endogenous mouse TSHR in transgenic TSHR/NOD.H2 h4 and nontransgenic NOD.H2 h4 mice. Quantitative real-time PCRs are shown as normalized fold expression relative to mouse b-actin of triplicates (mean + SD). Un, undetectable.

7 6 ATTEMPTED Ag-SPECIFIC THERAPY IN MURINE GRAVES DISEASE FIGURE 6. (A and B) htshr ELISA Abs in BALB/c mice injected three times with TSHR A-subunit protein and exposed to NaI water for 16 and 32 wk (A); Tg Abs in the same BALB/c mice (B, left panel) and in BALB/c mice that were not injected with TSHR A-subunit protein (B, right panel) after 16 and 32 wk on NaI water. Data are shown as OD 490 nm for individual mice (males and females). Dotted line for htshr ELISA Ab: mean + 2 SD for in nontransgenic NOD.H2 h4 mice; for TgAb: mean + 2 SD in BALB/c mice. (C E) NOD.H2 h4 mice injected three times with OVA Abs were tested for OVA Abs after 8 and 16 wk on NaI water (C). Data are shown as OD 290 nm for individual mice (males and females). Dotted line represents mean + 2 SD in NOD.H2 h4 mice on NaI for 16 wk. (D) Comparison of 16-wk values for htshr Ab ELISA OD values (mean + SE) in nontransgenic NOD.H2 h4 mice injected three times with htshr A-subunit protein (from mice shown in Fig. 2) versus mean OD values for nontransgenic NOD.H2 h4 mice injected three times with OVA [(values from (B)]. ELISA binding OD values for the mab used in each assay are included, namely (C) 3BD10 for htshr A-subunit protein (25) and (E) HYB for OVA (33). *p = (rank sum tests). of BALB/c mice were performed by injecting TSHR protein together with a variety of adjuvants (reviewed in Ref. 38). In contrast, heterologous OVA injected into NOD.H2 h4 mice led to a much lower specific Ab response in comparison with injection with inactive htshr A-subunit protein. These data point to the htshr A-subunit having unique antigenic properties rather than the NOD.H2 h4 mice being globally autoimmune prone or having multiple immune defects. In particular, NOD.H2 h4 mice do not develop autoantibodies to the endogenous mouse TSHR (20). The relatively low OVA Ab response versus the strong response to the htshr A-subunit in this mouse strain may relate to glycosylation, which plays a role in Ag uptake and internalization (e.g., Ref. 39). Thus, OVA has a single N-linked glycosylation site (32), whereas the htshr A-subunit has five N-linked glycosylation sites (40). Focusing on the outcome of injecting htshr A-subunit protein without adjuvant into nontransgenic NOD.H2 h4 and transgenic htshr/nod.h2 h4 mice, our data provide insight into the specificity of B cells in these two strains (Fig. 9). The injected htshr A-subunit protein will activate T cells in wild-type NOD.H2 h4 mice and stimulate memory T cells in the transgenics. T cells are critical for providing help to B cells to generate Abs. However, the outcome with respect to TSHR Abs depends on the B cell epitopes recognized. It should be emphasized that the two forms of TSHR A-subunits (active and inactive) have the identical amino acid composition and tertiary conformation but differ in their

8 The Journal of Immunology 7 FIGURE 7. TBI in female mice after 16 and 32 wk in (A) nontransgenic NOD.H2 h4 mice and (B) htshr transgenic NOD.H2 h4 mice. Data are shown as percentage inhibition for individual mice after three saline injections (white circles) or two injections (gray circles) or three injections (black circles) of htshr A-subunit protein in the absence of adjuvant. Dotted line represents the cutoff for nontransgenic NOD.H2 h4 mice (mean + 2 SD) at 16 wk. Values above this line are considered positive. *p = (t test). ns, not significant. quaternary structure (41). Evidence suggests that the former is a trimer and the latter a dimer, distinguished by differential recognition by pathogenic and nonpathogenic TSHR Abs (42). For this reason, we suggest that the htshr A-subunit expressed by the transgene (absent in the nontransgenics) influences the B cell precursor repertoire. We hypothesize that both transgenic and nontransgenic NOD.H2 h4 mice have precursor B cells for nonpathogenic ELISA-type TSHR Abs. In contrast, in htshr/nod.h2 h4 mice, the in vivo thyroidal expression of conformationally intact TSHR A-subunit by the transgene is responsible for selecting B cells specific for the epitopes of bioactive and pathogenic TSHR Abs. It is also possible that, as suggested for humans (43), intrathymic htshr A-subunit expressed in htshr/nod.h2 h4 mice plays a role in selecting pathogenic-type B cells. Injecting inactive TSHR A-subunit protein stimulates TSHR-specific T cells and B cell precursors for ELISA-type TSHR Abs in nontransgenics and, not surprisingly, expands these B cells in transgenic htshr/nod.h2 h4 mice. Remarkably, injecting inactive TSHR A-subunit protein, a related but incorrect protein, expands (with help from memory T cells) memory B cells specific for TBI/TSAb-type pathogenic TSHR Abs. B cells with affinity for self-ags (like the transgenic htshr) are tolerized by a number of mechanisms including receptor editing and anergy (functional unresponsiveness) rather than deletion as for self-reactive T cells (44). Using hen egg lysozyme-specific transgenic mouse models, Goodnow and colleagues (45) demonstrated that self-reactive B cells were not eliminated when this Ag was expressed by thyroid cells. Similarly, in our spontaneous model, B cells specific for the intrathyroidal htshr A-subunit remain in the repertoire and can be expanded by the crossreacting Ag, the inactive htshr A-subunit. Our observations have important implications for Ag-specific immunotherapeutic approaches to deviate pathogenic TSHR Abs using an inappropriate form of htshr A-subunit protein. It is likely that Graves patients cannot be treated by injecting inactive TSHR A-subunit protein because, as in htshr/nod.h2 h4 mice, stimulation of memory T cells and B cells specific for pathogenic Abs will lead to exacerbation of hyperthyroidism. Further, Graves orbitopathy involves both TSHR Abs and cytokines, the latter secreted in response to TSHR-specific T cell activation (reviewed in Ref. 46). For these reasons, injecting inactive htshr A-subunit protein is precluded as an Ag-specific treatment to divert production of pathogenic Abs and alleviate hyperthyroidism in Graves disease. FIGURE 8. TSAb activity in females after 16 and 32 wk in (A) nontransgenic NOD.H2 h4 mice and (B) htshr transgenic NOD.H2 h4 mice. Data are shown as % control values for individual mice. Saline injections are shown as white circles; htshr A-subunit protein injected twice (gray circles) or three times (black circles) in the absence of adjuvant. Dotted line represents the cutoff for nontransgenic NOD.H2 h4 mice at 16 wk. Values above this line are considered positive. *p = (t test). ns, not significant.

9 8 ATTEMPTED Ag-SPECIFIC THERAPY IN MURINE GRAVES DISEASE FIGURE 9. Schematic representation of likely B cell precursors and their expansion in (A) nontransgenic NOD.H2 h4 mice and (B) htshr transgenic NOD.H2 h4 mice in response to injecting htshr A-subunit protein (inactive form) without adjuvant. Not shown are Ag-specific memory T cells in htshr transgenic NOD.H2 h4 mice that will be activated by the injected htshr A-subunit protein and provide critical help for Ab production by B cells. Acknowledgments We thank Dr. Jean Ruf (INSERM-URA, Faculté de Médecine, Marseille, France) for generously providing mouse mabs to human TPO. Disclosures The authors have no financial conflicts of interest. References 1. Rapoport, B., G. D. Chazenbalk, J. C. Jaume, and S. M. McLachlan The thyrotropin (TSH) receptor: interaction with TSH and autoantibodies. Endocr. Rev. 19: Burch, H. B., K. D. Burman, and D. S. Cooper A 2011 survey of clinical practice patterns in the management of Graves disease. J. Clin. Endocrinol. Metab. 97: Burch, H. B., and D. S. Cooper Management of Graves Disease: a review. JAMA 314: Neumann, S., E. A. Nir, E. Eliseeva, W. Huang, J. Marugan, J. Xiao, A. E. Dulcey, and M. C. Gershengorn A selective TSH receptor antagonist inhibits stimulation of thyroid function in female mice. Endocrinology 155: Davies, T. F., and R. Latif Targeting the thyroid-stimulating hormone receptor with small molecule ligands and antibodies. Expert Opin. Ther. Targets 19: Furmaniak, J., J. Sanders, and B. Rees Smith Blocking type TSH receptor antibodies. Auto Immun Highlights 4: Stan, M. N., J. A. Garrity, B. G. Carranza Leon, T. Prabin, E. A. Bradley, and R. S. Bahn Randomized controlled trial of rituximab in patients with Graves orbitopathy. J. Clin. Endocrinol. Metab. 100: Salvi, M., G. Vannucchi, N. Currò, I. Campi, D. Covelli, D. Dazzi, S. Simonetta, C. Guastella, L. Pignataro, S. Avignone, and P. Beck-Peccoz Efficacy of B-cell targeted therapy with rituximab in patients with active moderate to severe Graves orbitopathy: a randomized controlled study. J. Clin. Endocrinol. Metab. 100: Steinman, L The road not taken: antigen-specific therapy and neuroinflammatory disease. JAMA Neurol. 70: Roep, B. O., and M. Peakman Antigen targets of type 1 diabetes autoimmunity. Cold Spring Harb. Perspect. Med. 2: a Available at: perspectivesinmedicine.cshlp.org/content/2/4/a abstract?sid=dc1e8a0e-23af aede-99c3d19571be. 11. Hollowell, J. G., N. W. Staehling, W. D. Flanders, W. H. Hannon, E. W. Gunter, C. A. Spencer, and L. E. Braverman Serum TSH, T(4), and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J. Clin. Endocrinol. Metab. 87: Kita, M., L. Ahmad, R. C. Marians, H. Vlase, P. Unger, P. N. Graves, and T. F. Davies Regulation and transfer of a murine model of thyrotropin receptor antibody mediated Graves disease. Endocrinology 140: Nagayama, Y., H. Mizuguchi, T. Hayakawa, M. Niwa, S. M. McLachlan, and B. Rapoport Prevention of autoantibody-mediated Graves -like hyperthyroidism in mice with IL-4, a Th2 cytokine. J. Immunol. 170: Nagayama, Y., K. Watanabe, M. Niwa, S. M. McLachlan, and B. Rapoport Schistosoma mansoni and alpha-galactosylceramide: prophylactic effect of Th1 Immune suppression in a mouse model of Graves hyperthyroidism. J. Immunol. 173: Gilbert, J. A., S. L. Kalled, J. Moorhead, D. M. Hess, P. Rennert, Z. Li, M. Z. Khan, and J. P. Banga Treatment of autoimmune hyperthyroidism in a murine model of Graves disease with tumor necrosis factor-family ligand inhibitors suggests a key role for B cell activating factor in disease pathology. Endocrinology 147:

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