Cepharanthine blocks TSH receptor peptide presentation by HLA-DR3: Therapeutic implications to Graves’ disease

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Cepharanthine blocks TSH receptor peptide presentation by HLA-DR3: Therapeutic implications to Graves’ disease☆ Cheuk Wun Lia, Roman Osmanb, Francesca Menconic, Erlinda Concepciona, Yaron Tomera,∗ a

Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA c Endocrinology Unit, University of Pisa, Pisa, Italy b

ARTICLE INFO

ABSTRACT

Keywords: Graves' disease T-cells TSH receptor Thyroid Autoimmunity

We have previously identified a signature HLA-DR3 pocket variant, designated HLA-DRβ1-Arg74 that confers a high risk for Graves' Disease (GD). In view of the key role of HLA-DRβ1-Arg74 in triggering GD we hypothesized that thyroid-stimulating hormone receptor (TSHR) peptides that bind to the HLA-DRβ1-Arg74 pocket with high affinity represent key pathogenic TSHR peptides triggering GD, and that blocking their presentation to CD4+ Tcells can be used as a novel therapeutic approach in GD. There were several previous attempts to identify the major pathogenic TSHR peptide utilizing different methodologies, however the results were inconsistent and inconclusive. Therefore, the aim of our study was to use TSHR peptide binding affinity to HLA-DRβ1-Arg74 as a method to identify the key pathogenic TSHR peptides that trigger GD. Using virtual screening and ELISA and cellular binding assays we identified 2 TSHR peptides that bound with high affinity to HLA-DRβ1-Arg74 TSHR.132 and TSHR.197. Peptide immunization studies in humanized DR3 mice showed that only TSHR.132, but not TSHR.197, induced autoreactive T-cell proliferation and cytokine responses. Next, we induced experimental autoimmune Graves’ disease (EAGD) in a novel BALB/c-DR3 humanized mouse model we created and confirmed TSHR.132 as a major DRβ1-Arg74 binding peptide triggering GD in our mouse model. Furthermore, we demonstrated that Cepharanthine, a compound we have previously identified as DRβ1-Arg74 blocker, could block the presentation and T-cell responses to TSHR.132 in the EAGD model.

1. Introduction Graves’ disease (GD) is an autoantibody-mediated autoimmune disease that is characterized clinically by hyperthyroidism and pathologically by infiltration of thyroid by T and B cells reactive to the thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase (TPO), and thyroglobulin (Tg) in most patients. GD is caused by direct stimulation of thyroid epithelial cells by TSHR antibodies, triggering signaling cascades within thyrocytes that lead to over-production and secretion of thyroid hormones resulting in clinical hyperthyroidism [1]. The etiology of GD is believed to involve an interplay between susceptibility genes and environmental factors that modulate susceptibility gene function through epigenetic marks, thereby triggering disease [2–4]. While several susceptibility genes for GD have been mapped, HLADR3 and TSHR are the most important genes showing linkage and association with GD [5,6]. In view of its importance to the etiology of GD

our lab has sequenced the HLA-DR3 gene in order to pinpoint the exact DR3 peptide-binding pocket sequence and structure that triggers GD. Using this approach we have identified a specific HLA-DR pocket sequence that is strongly associated with both Graves' disease (GD) and Hashimoto's thyroiditis (HT) [5,7]. The key amino acid that confers risk for GD is arginine at position 74 of the HLA-DRβ1 chain (this genetic variant is designated HLA-DRβ1-Arg74) [5]. The A subunit of the extracellular domain of TSHR is the major autoantigen in GD, mediating the T- and B-cell immune responses that cause GD [8,9]. The sequence of events leading to GD start when pathogenic TSHR peptides are presented by HLA class II on antigen presenting cells to CD4+ T-cells. CD4+ T-cells then recognize the HLA class II-TSHR peptide complex and initiate immune responses, including signals for B-cell proliferation and differentiation into plasma cells, which produce and secrete anti-TSHR stimulating antibodies [1]. There were several attempts in previous studies to identify the key pathogenic TSHR peptides triggering GD. However, their results were

This work was supported in part by NIDDK (grant numbers DK067555 & DK073681, to YT). Corresponding author. 1300 Morris Park Ave, Bronx, NY, 10461, USA. E-mail address: [email protected] (Y. Tomer).

☆ ∗

https://doi.org/10.1016/j.jaut.2020.102402 Received 25 October 2019; Received in revised form 26 December 2019; Accepted 1 January 2020 0896-8411/ © 2020 Elsevier Ltd. All rights reserved.

Please cite this article as: Cheuk Wun Li, et al., Journal of Autoimmunity, https://doi.org/10.1016/j.jaut.2020.102402

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inconsistent and inconclusive and it is still unclear which TSHR peptides are the major pathogenic peptides that are critical to triggering the autoimmune responses in GD [10–21]. The inconsistencies between previous studies could be due to several factors including heterogeneity of HLA-DR alleles among the patients tested and the different methodologies and approaches employed. But most importantly, previous studies tested T-cell responses in patients with established GD that may have experienced epitope spreading, and therefore the initial triggering epitope may not have elicited T-cell responses at the time the studies were conducted. Indeed, there is abundant evidence for epitope spreading in GD [12,14], and there is evidence of B-cell epitope spreading when BALB/c or CBA/J mice were immunized with TSHR ECD [22]. To avoid these impediments we used a novel strategy to identify the key triggering pathogenic peptides in GD before epitope spreading occurred. Our strategy was based on identifying pathogenic peptides that can be most efficiently presented to CD4+ T-cells by HLA-DRβ1-Arg74, shown to be the key HLA class II allele associated with GD. We hypothesized that pathogenic TSHR peptides that trigger GD must bind specifically and with high affinity to HLA-DRβ1-Arg74. Using this strategy we report here on the identification of a major TSHR pathogenic peptide that triggers GD. Moreover, we show that, Cepharanthine, a small molecule we previously reported to block DRβ1-Arg74 [23], can block autoreactive T-cell activation by TSHR in a mouse model of GD.

incubated protein-peptide complex were added onto the plate and shaken at slow speed for 2 h at room temperature. After washing for 4 times, DELFIA Europium-labeled streptavidin (PerkinElmer) diluted in DELFIA assay buffer (PerkinElmer) was added for 30 min and shaken at slow speed at room temperature. After washing for 6 times, DELFIA Enhancement Solution (PerkinElmer) was added for 1 h or until the optimal signal was reached. Time-resolved fluorescence was measured using a BMG reader (BMG Labtech, Cary, NC). The experiment was performed in triplicates. As negative control we added biotinylated TSHR peptide that was not pre-incubated with HLA-DRβ1-Arg74. Fold increase of was calculated as follows: [HLA-DRβ1-Arg74 protein + peptide/peptide alone]. Peptides binding with greater than 2 fold (>10 standard deviations over the average for peptide alone with no HLA) was considered as positive. 2.3. Cell culture VAVY cells that are homozygotes for HLA-DR3 and positive for HLA-DRβ1-Arg74 (European Collection of Authenticated Cell Cultures) were cultured as previously described [23]. Briefly, VAVY cells were cultured in RPMI (ATCC, Manassas, VA) supplied with 10% FBS (SigmaAldrich, St. Louis, MO), 1% penicillin-streptomycin (Corning, NY), 2 mM glutamine (Corning, NY) and 0.01 mg/ml of ciprofloxacin hydrochloride (Bioworld, Dublin, OH). Cells were grown at 37 °C, 5% CO2 and passaged 1–2 times a week.

2. Materials and Methods

2.4. Analysis of peptide binding by flow cytometry

2.1. Virtual screen of TSHR peptides First, we performed a virtual screen to identify candidate TSHR peptides that can bind with high affinity to HLA-DRβ1-Arg74. We used the allele DRB1*0301 which has Arg at position 74. We also ran a virtual screen on the DRB1*0317 allele, which has a Gln in the place of Arg. We then compared the predicted potency of epitopes for DRB1*0301 with the predicted potency for DRB1*0317 for the same sequences. The virtual screen of the ECD of the TSHR was done with the NetMHCIIpan 3.1 server [24]. The NetMHCIIpan approach is based on multiple artificial neural networks trained on experimentally measured binding data to a representative set of MHC II molecules. This approach has an improved predictive power for the core 9-mer sequence.

Peptide binding to HLA-DRβ1-Arg74 positive VAVY cells was performed similar to previously described [23]. Briefly, N-terminal biotinylated peptides were used for testing binding to VAVY cells that express homozygous HLA-DR3 on their surface (HLA-DRβ1-Arg74 positive). A scrambled thyroglobulin peptide (scr2098, HDLFSRIDSSVVVVP) was used as a negative control. VAVY cells were seeded at 2.5 × 106 cells/ml in a 24-well plate (BD Bioscience). Increasing doses (10 μM, 20 μM, 50 μM) of biotinylated peptide was incubated with VAVY cells for 24 h. 5 μg/ml APC streptavidin (BD Biosciences, Franklin Lakes, NJ) was used to detect binding of the peptide to VAVY cells. 5 μg/ml L243 (described above) and donkey anti-mouse IgG-PE (1:100) (abcam, Cambridge, UK) were used to detect DR3 expression.

2.2. In vitro screening of TSHR peptides

2.5. Mice

To complement the virtual screen we also performed a biochemical screen using a unique ELISA we developed for screening peptides that bind with high affinity to HLA-DRβ1-Arg74. The forty-three TSHR peptides tested in the in vitro screening (Supplementary Table 1) were synthesized (Genscript, Piscataway, NJ) and tested for binding to the HLA-DRβ1-Arg74 pocket using an immunoassay we previously described [23,25]. These 43 peptides included the ones we identified by the virtual screen with predicted KD < 15,000 nM, regions that were not covered by the virtual screen, and some peptides reported in literature [12–14]. Briefly, 0.012 mg/ml of HLA-DRβ1-Arg74 protein was incubated with 10 μM biotinylated TSHR peptides (Genscript) for 48 h at 37 °C in binding buffer (0.1% BSA/PBS with 0.05% Triton (PBST), Sigma Aldrich). On the day before the immunoassay was performed, a 96-well DELFIA yellow plate (PerkinElmer Life Sciences) was coated overnight with 20 μg/ml of L243 antibody [Hybridoma was purchased from ATCC, catalog number HB-55, and IgG was purified by QED Biosciences (San Diego, CA)] in bicarbonate buffer, pH 9.6 (SigmaAldrich). L243 is a monoclonal antibody that specifically recognizes the DRα chain when it is properly folded and complexed with the β chain [26]. The plate was then washed with DELFIA wash buffer (diluted 1:25 from DELFIA wash concentrate, PerkinElmer) to wash off the excess L243 antibody. Blocking was done using 2.5% BSA in PBS at room temperature for 1 h. After washing for 4 times, 100 μl of the pre-

2.5.1. NOD-DR3 “humanized” mice These mice are knockout for murine MHC class II and express DRB1*0301 (confirmed by sequencing to be HLA-DRβ1-Arg74 positive); they were used to confirm that identified TSHR peptides induce Tcell responses. The derivation and genotyping of the mice was described previously [23]. 2.5.2. BALB/c-DR3 “humanized” mice We used novel humanized BALB/c-DR3 transgenic mice that we generated as follows: C57BL/6-DR3 mice (null for murine class II MHC and expressing human DR3 harboring DRβ1-Arg74) were obtained from Dr. Chella David (Mayo Clinic) and crossed with wild type BALB/c mice (The Jackson Laboratories) since the BALB/c background is most susceptible to EAGD [27]. The F1 progeny (50% BALB/c background, inheriting one copy of murine class II MHC) were crossed with each other and genotyped; approximately 25% were both positive for human DR3 and knockout of murine MHC class II. These F2 progeny (DR3positive and null of murine MHC II) were back-crossed with wild type BALB/c mice for one more cycle. The resulting F3 progeny (receiving one copy of murine MHC II from wild type BALB/c mice) were again crossed with each other and their F4 progeny were genotyped to select for the ~25% that both expressed human DR3 and were KO for murine MHC II. These F4 mice were bred into a colony and used in our 2

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experiments (designated BALB/c-DR3 mice from here on).

temperature. After three more washes, substrate was added for 20 min in the dark at room temperature. Stop solution was added and absorbance was read at 450 nm using a BMG ELISA reader (BMG Labtech, Cary, NC). Due to a switch to an updated version of the kit that happened while we were working on this project (the updated kit uses a different antibody to recognize TSHR), the values obtained were normalized between the old and updated kits by dividing the sample results by the positive cut-off reference value (0.4 IU/l in the old version [catalog no. 3805], 1.5 IU/l in the updated version [catalog no. 3505]). The fold increase (reading obtained divided by the positive cut-off reference value) was used to compare the change in TRAb levels.

2.6. Genotyping murine class II MHC knockout mice Genotyping to confirm knockout of the murine class II MHC was performed in the F2 and F4 progeny described above. Briefly, mouse tails were clipped and collected in Trizol reagent (Life Technologies, Carlsbad, CA). RNA extraction was performed, followed by reverse transcription using SuperScript III first-strand synthesis system for RTPCR (Life Technologies). 1–2 μg RNA was reversed transcribed to cDNA. The I-A alpha chain was detected by forward primer 5′ CACAG ACGGCGTTTATGAGA 3′ and reverse primer 5′ GGCACACACCACAGT TTCTG 3’. The I-A beta chain was detected by forward primer 5′ CAA CCACCACAACACTCTGG 3′ and reverse primer 5′ ACAGTGATGGGGC TCTTCAG 3’. The absence of both chains indicates knockout of murine class II MHC.

2.12. Testing stimulating activity of TSHR antibodies in mouse sera We measured the stimulating activity of TSHR antibodies in mouse sera by testing their ability to induce cAMP production in cells expressing the TSHR. A stable cell line of CHO-HA-TSHR luciferase cells was generously provided by Drs. Rauf Latif and Terry Davies [30]. Cells were grown in Ham's F12 medium (Gibco, Gaithersburg, MD) supplied with 10% FBS (Sigma-Aldrich, St. Louis, MO), 1× penicillin-streptomycin (Corning, NY) and 100 μg/ml of hygromycin (Invitrogen, Carlsbad, CA). CHO-HA-TSHR luciferase cells (500,000 cells/ml) were seeded in a 96-well plate and incubated for 24 h at 37 °C. Bovine TSH (Sigma-Aldrich, catalog no. T8931) at concentrations of 1 μU/ml to 10,000 μU/ml was added to generate a dose response curve. Sera from BALB/c-DR3 mice immunized with AdTSHR (n = 14) or AdLacZ (n = 12) were diluted 1:2 in Ham's F12 medium. 50 μl of increasing concentrations of TSH and diluted serum samples were added to cells and incubated for 5 h at 37 °C. After 5 h incubation, 50 μl of Bright-Glo reagent (Promega, WI, catalog no. E2610) were added to each well. Followed by 2–3 min gentle shaking, luciferase activity was measured using a BMG ELISA reader (BMG Labtech, Cary, NC).

2.7. Peptide immunization Female NOD-DR3 mice, 4–6 weeks old, were injected subcutaneously with the peptides tested in this study in Complete Freund Adjuvant (CFA) (Sigma-Aldrich) to induce T-cell response as previously described [23,25]. Mice were immunized with peptides on day 0 and boosted on day 7, then sacrificed on day 21. Nine mice were immunized with TSHR.132 and eight mice were immunized with TSHR.197. 2.8. Lymphocytes isolation Spleen and draining lymph nodes were collected from mice upon sacrifice. Lymphocytes were isolated as previously described [23,25]. 2.9. T-cell proliferation and cytokine activation analyses Cells isolated from the spleen and lymph nodes and were tested for proliferative and cytokine responses to TSHR peptides as previously described [23,25].

2.13. Free T4 measurements Sera collected from mice were assayed for free thyroxine (fT4) measurements using free thyroxine ELISA kit (Alpha Diagnostic International, San Antonio, TX, catalog no. 1110). Assay was performed according to manufacturer's protocol. The standards were run simultaneously with the serum samples to obtain a standard curve. Briefly, serum samples were added to wells pre-coated with T4-specific antibodies. Diluted enzyme conjugate were added for 1 h at 37 °C, followed by three washes using wash buffer supplied in the kit. TMB substrate was added for 15 min at 37 °C, followed by stop solution. Absorbance at 450 nm was measured using a BMG ELISA reader (BMG Labtech, Cary, NC).

2.10. Induction of experimental autoimmune Graves’ disease (EAGD) in DR3 “humanized” mice EAGD was induced by cDNA immunization according to the Nagayama model [28] with the modifications by Rapoport and McLachlan [9,29]. Adenoviral vectors containing AdTSHR-289 (A-subunit) or AdLacZ control cDNA were generously provided to us by Dr. Rapoport and Dr. McLachlan (Cedars Sinai Medical Center, Los Angeles, CA), and propagated by ViraQuest Inc. (North Liberty, Iowa). Stock solutions contained 1.1 × 1012 particles/ml. 5 × 109 particles were injected intramuscularly in 50 μl diluted in 10% glycerol (Sigma-Aldrich) in PBS in the thigh muscle at week 0, week 3 and week 6. Mice were sacrificed at week 9. Spleen, thyroid and serum were collected from each mouse at sacrifice.

2.14. T3 measurements Sera collected from mice were assayed for T3 measurements using Total Triiodothyronine (total T3) ELISA kit (Alpha Diagnostic International, San Antonio, TX, catalog no. 1700). Assay was performed according to manufacturer's protocol. The standards were run simultaneously with the serum samples to obtain a standard curve. Briefly, serum samples were added to wells pre-coated with T3-specific antibodies. Diluted enzyme conjugate was added for 1 h at room temperature with shaking, followed by three washes with wash buffer supplied in the kit. TMB substrate was added for 15 min at room temperature, followed by stop solution. Absorbance at 450 nm was measured using a BMG ELISA reader (BMG Labtech, Cary, NC).

2.11. TRAb measurements Sera collected from mice were assayed for TSHR receptor antibodies using Medizym T.R.A. human ELISA kit (Medipan GMBH, Berlin, Germany, catalog no. 3505). The assay was performed according to manufacturer's protocol. Calibrators that were run simultaneously with serum samples were used to obtain a standard curve for determining sample concentrations. Briefly, standards and serum samples diluted in incubation buffer were added to plates pre-coated with human recombinant TSH receptor for 2 h at room temperature with shaking. Plates were then washed with washing solution supplied in the kit, and TRA-biotin (a biotinylated tracer antibody that binds to the free epitopes of the receptor) was added for 20 min at room temperature. After another wash, diluted conjugate was added for 20 min at room

2.15. Histology Thyroid glands were dissected and removed from BALB/c-DR3 mice after sacrifice and stored in 10% formalin until processing. After creating paraffin blocks, thyroids were sectioned and stained with 3

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hematoxylin-eosin for histological analysis (Histology and Comparative Pathology Core, Albert Einstein College of Medicine). Thyroid sections slides were scanned by 3DHistec Pannoramic 250 Flash II slide scanner (Analytical Imaging Facility, Albert Einstein College of Medicine).

Table 1 Synthesized TSHR peptides used for testing binding to HLA-DRβ1-Arg74.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

2.16. Determining potency of Cepharanthine in blocking TSHR peptide binding Cepharanthine (Microsource Discovery Systems, Gaylordsville, CT) is a small molecule previously identified by us as an HLA-DRβ1-Arg74 blocker [23]. Percent inhibition of TSHR peptide binding was tested at decreasing concentrations of Cepharanthine. Cepharanthine was serially diluted to 0.0125 mM final concentration and incubated with the HLA-DRβ1-Arg74–TSHR peptide complex for 48 h at 37 °C for assessing the percent inhibition. Immunoassay was performed with the modification as described in section 2.2, except that Cepharanthine was incubated with the HLA-DRβ1-Arg74–TSHR peptide complex. Percent inhibition was calculated by the following formula: 100–100 × (HLADRβ1-Arg74-TSHR peptide-small molecule/HLA-DRβ1-Arg74-TSHR peptide (no small molecule)).

Peptide

Sequence

Predicted KD (nM)

TSHR80-94 TSHR53-67 TSHR197-216 TSHR54-68 TSHR78-94 TSHR132-150 TSHR202-216 TSHR137-150 TSHR137-151 TSHR266-280 TSHR266-284 TSHR267-281 TSHR246-266 TSHR211-225 TSHR51-65

RIYVSIDVTLQQLES STQTLKLIETHLRTI FNGTKLDAVYLNKNKYLTVI TQTLKLIETHLRTIP ISRIYVSIDVTLQQLES GIFNTGLKMFPDLTKVYST LDAVYLNKNKYLTVI GLKMFPDLTKVYST GLKMFPDLTKVYSTD SLSFLHLTRADLSYP SLSFLHLTRADLSYPSHCC LSFLHLTRADLSYPS LEHLKELIARNTWTLKKLPLS KYLTVIDKDAFGGVY PPSTQTLKLIETHLR

93.1 151.5 154.5 166.5 168.9 202.6 222.2 236.0 236.0 259.3 259.3 261.8 307.4 462.7 486.7

out according to the guidelines of the IACUC of the Icahn School of Medicine at Mount Sinai and Albert Einstein College of Medicine.

2.17. Cepharanthine inhibition of EAGD ex vivo

3. Results

Splenocytes were incubated with 1 μM Cepharanthine together with TSHR.132 (20 μg/ml) to block antigen presentation in the EAGD model. To demonstrate that the inhibition is specific, DMSO was used as a control. T-cell activation and cytokine production were analyzed as described above.

3.1. Virtual screening of TSHR peptides Virtual screening of the ECD of the TSHR was conducted with the NetMHCIIpan 3.1 server algorithm. Each peptide of a given length was evaluated for its ability to bind to the MHC molecule based on the 9mer core sequence (the peptide register). Since the training of the network was done on various length peptides, some sequences show differences depending on the flanking residues on both ends of the register. The initial screen was done on sequences of 15 amino acids. The virtual screen predicted that some peptides will bind with varying affinities, but most peptides were predicted not to bind at all. This initial survey identified 15 peptides with high binding affinity to HLADRβ1-Arg74 (i.e. predicted KD of <500 nM, Table 1).

2.18. Molecular dynamics (MD) simulation of DR3 in complex with TSHR peptides The core sequence in the TSHR peptide was adopted from the analysis with the NetMCHIIpan server. The backbone of the peptides including the flanking residues was superimposed on the complex of DR3 with the CLIP peptide (PDB: 1A6A) [31]. The complex was used as an initial structure to build the system for simulations with AMBER [32]. The complex was placed in a periodic box and filled with TIP3P waters and ions at an effective concentration of 0.15 M. The system was minimized and heated to 300 K while restraining the protein with a force constant of 10 kcal/mol/Å2. The restraints were gradually released during an equilibration phase. The equilibrated system was simulated at NPT conditions for 200 ns (20,000 frames). The trajectories were analyzed using the AmberTools package and the MMGB(PB)/SA analysis was conducted with the same program. Briefly, the MMGB (PB)/SA method estimates the binding energy as a sum of molecular mechanical interaction terms (MM) and the change in solvation is obtained from a Generalized Born (GB) or Poisson-Boltzmann (PB) evaluation of the electrostatic components and the Surface Area (SA) for the non-polar contributions. The entropy is estimated by a Normal Mode approach. The total interaction energy can be decomposed into contributions from individual residues [33].

3.2. In vitro binding of TSHR peptides to HLA-DRβ1-Arg74 We tested forty-three TSHR peptides for binding to HLA-DRβ1Arg74 protein utilizing the unique in vitro binding assay we developed (see Materials and Methods). The tested TSHR peptides included those showing at least low binding affinity (i.e. predicted KD < 15,000 nM) in the virtual screen, those reported in the literature [12–14], and those that were derived from TSHR regions not covered in the virtual screen in order to ensure increased coverage and density (Supplementary Table 1). Our in vitro binding results showed that two TSHR peptides bound HLA-DRβ1-Arg74 with high affinity - TSHR.132–150 (designated TSHR.132 from here on) and TSHR.197–216 (designated TSHR.197 from here on) (Fig. 1). Both of these peptides were among the top 15 peptide binders based on the virtual screen and had predicted binding affinities of 154.5 nM (TSHR.197) and 202.6 nM (TSHR.132). Other peptides with predicted KD > 500 nM did not bind in the ELISA demonstrating a reasonable predictive power (2 out of 15) of the virtual screen to reduce the number of in vitro experiments (data not shown). To test whether the two peptides (TSHR.132 and TSHR.197) have unique selectivity for HLA-DRβ1-Arg74 we conducted a virtual screen on another subtype of HLA-DR3, HLA-DRB1*0317 that contains a Gln in position 74 of the beta chain (the protective HLA-DR allele in GD). TSHR.132 and TSHR.197 were predicted to be of substantially lower affinity to HLA-DRB1*0317: 1052.7 and 436.3 nM, respectively. In view of their predicted selectivity to the HLA-DRβ1-Arg74 variant of HLA-DR3 TSHR.132 and TSHR.197 could be potentially pathogenic TSHR peptides that can be presented by HLA-DRβ1-Arg74 to trigger GD

2.19. Statistical analysis Prism 5 software was used to perform the statistical analysis. Student's t-test (unpaired t-test, one-tailed) was used for comparisons of means of the experimental vs. control groups for each of the continuous variables measured. A p value < 0.05 was considered statistically significant. 2.20. Study approval The project was approved by the Institutional Animal Care and Use Committees (IACUC) of the Icahn School of Medicine at Mount Sinai and Albert Einstein College of Medicine. All animal studies were carried 4

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Lys71β (see Fig. 2 and Table 2). In TSHR.132 the hydrophobic Met in position 1 matches the P1 pocket better than the Thr in TSHR.197; even though these interactions are moderate they differ by nearly 5 kcal/ mol. In position 9 the Tyr in TSHR.132 also interacts better than the Asn in TSHR.197 by more than 6 kcal/mol. This is mostly due to a better Hbond with Asp57β. Residue 6 often plays an important role as well and here the contribution from Thr in TSHR.132 is nearly the same as that of the Val in TSHR.197. Considering the contributions from these four residues the decomposition provides a rationalization, albeit incomplete, for the better binding of TSHR.132 than of TSHR.197. 3.4. TSHR.132 and TSHR.197 bind to VAVY cells in a dose-dependent manner

Fig. 1. Result of the in vitro screening of TSHR peptides binding to HLA-DRβ1Arg74. Two out of 15 TSHR peptides with predicted KD < 500 nM bind in vitro to HLA-DRβ1-Arg74. TSHR.132 and TSHR.197 showed significant binding to HLA-DRβ1-Arg74. The other 13 peptides predicted to bind did not show strong binding. Time-resolved fluorescence was measured. Fold increase was calculated by (HLA-DRβ1-Arg74 + TSHR peptide/TSHR peptide alone). Details described in Materials and Methods.

Increasing concentrations (10 μM, 20 μM, 50 μM) of biotinylated TSHR.132 and TSHR.197 were incubated with VAVY B-cell line (homozygous for HLA-DRβ1-Arg74) for 24 h. Percentage of DRβ-Argpositive cells were detected by L243 monoclonal anti-DR antibody and donkey anti-mouse IgG-PE. Percent of peptide binding was detected by APC-Streptavidin. TSHR.132 bound to DRβ-Arg74-positive cells in a dose-dependent manner, with binding increasing from 5.1% (10 μM, Fig. 3A), to 21.7% (20 μM, Fig. 3B) and 51% (50 μM, Fig. 3C). Similarly, TSHR.197 bound to DRβ-Arg-positive cells dose-dependently, with binding increasing from 14.7% (10 μM, Fig. 3D), to 19.4% (20 μM, Fig. 3E) and 72.9% (50 μM, Fig. 3F). The negative control scrambled thyroglobulin peptide (scr2098, Fig. 3G) only showed 5% binding to DRβ-Arg74-positive cells at 50 μM, confirming that the binding of TSHR.132 and TSHR.197 to VAVY cells was specific.

but cannot be presented by the protective HLA-DR allele. 3.3. Molecular dynamic simulations Considering that the NetMHCIIpan predicted affinities are based on a neural network rather than a physical model, we decided to explore the contributions to binding by performing molecular dynamic (MD) simulations. Using the results from the MD simulations we attempted to compute the binding affinity of the peptides. The binding free energy estimated by the MM-GBSA approach from a single trajectory of the complex proved to yield binding energies that were much too negative with large error estimates (−41 ± 12 or −34 ± 17 kcal/mol). However, it is well known that the calculation of the change in entropy of binding is underestimated because the entropy of the free peptide cannot account for the multiplicity of states it can occupy. Therefore, we estimated the contribution to binding of individual residues by decomposing the interaction energy. The results for TSHR.132 and TSHR.197 are shown in Table 2. The three most important residues that contribute to binding are in positions 1, 4 and 9. Fig. 2 depicts the interaction energies mapped onto the surface of the interacting residues in HLA-DRβ1-Arg74. The energies range from 0 (blue) to −18 (red) kcal/mol. Both peptides share the characteristic Asp in position 4, which interacts with the DRβ1-Arg74 and anchors the peptide to the MHC groove. Asp (4) in TSHR.132 makes a considerably larger contribution because of a better interaction with DRβ1-Arg74 as well as

3.5. Only TSHR.132 induces T-cell immune responses in NOD-DR3 mice 3.5.1. T-cell proliferative responses Splenocytes and lymph node cells isolated from TSHR.132 or TSHR.197 peptide-immunized NOD-DR3 mice were labeled with CFSE and tested for proliferative responses after 5 days of incubation with: 1) TSHR.132 and TSHR.197, the two peptides that bound with high affinity to HLA-DRβ1-Arg74 in vitro; 2) APO peptide (IPDNLFLKSDGRIKYTLNK, negative control); or 3) anti-mouse CD3/CD28 beads (positive control). Stimulation index ≥2 was considered positive. TSHR.132 stimulated T-cells of mice immunized with TSHR.132 (n = 9), with stimulation index of 3.67 (Fig. 4A). In contrast, TSHR.197 did not stimulate T-cells of mice immunized with TSHR.197 (n = 8, stimulation index = 1.13, Fig. 4B). 3.5.2. Cytokine production Splenocytes and lymph node cells isolated from peptide-immunized NOD-DR3 mice were incubated with the same stimulants for 48 h to test for cytokine responses. Stimulants included 1) TSHR.132 and TSHR.197; 2) APO peptide (negative control); or 3) anti-mouse CD3/ CD28 beads (positive control). Lymphocytes isolated from mice immunized with TSHR.132 showed significant Th1 cytokine responses to TSHR.132 including interferon gamma (IFN-g) (470 pg/ml, Fig. 4C) and IL-2 (49.2 pg/ml, Fig. 4D), but there was no production of the Th2 cytokine IL-4 as well as IL-10 (Fig. 4E and F). In contrast, lymphocytes isolated from mice immunized with TSHR.197 showed no increase in cytokine production when splenocytes were stimulated with TSHR.197 (Fig. 4G–J). These data demonstrated that even though both peptides bound with high affinity to DRβ1-Arg74 only TSHR.132 was able to activate T-cell responses.

Table 2 Residue-based decomposition of peptide-DR3 interaction energy. TSHR.132

TSHR.197

Residue

Int. Energy (kcal/mol)

Residue

Int. Energy (kcal/mol)

Thr Gly Leu Lys Met (1) Phe (2) Pro (3) Asp (4) Leu (5) Thr (6) Lys (7) Val (8) Tyr (9) Ser Thr

1.69 −0.63 −3.08 −4.88 −9.76 −4.73 −5.45 −9.98 −5.22 −3.68 3.18 −4.72 −8.74 −5.79 −2.38

Ala Phe Asn Gly Thr (1) Lys (2) Leu (3) Asp (4) Ala (5) Val (6) Tyr (7) Leu (8) Asn (9) Lys Asn

0.75 −3.44 −2.48 −2.15 −4.96 −3.43 −6.59 −6.40 −1.50 −3.22 −5.05 −4.95 −2.40 −0.56 −0.22

3.6. T-cell responses to TSHR peptides in BALB/c-DR3 mice induced with EAGD EAGD was induced in BALB/c-DR3 “humanized” mice by cDNA immunization with adenovirus expressing the A-subunit of TSHR (AdTSHR) or with adenovirus expressing the LacZ gene (AdLacZ) 5

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Fig. 2. Interaction energies between P1, P4 and P9 of TSHR peptides and the residues of the DRβ1-Arg74 mapped on the surface of the protein. The range of the interaction is depicted by the slide rule and is coded from 0 (blue) through −9 (white) to −18 (red) - −18 kcal/mol. Note the intense red for Arg74 and the adjacent Lys71.

Fig. 3. Results of TSHR peptide binding to HLA-DRβ1-Arg74 in a cell-based assay using flow cytometry. Biotinylated TSHR.132 binds to DRβ1-Arg74-positive cells at different doses-10 μM (A), 20 μM (B) or 50 μM (C). Similarly, biotinylated TSHR.197 binds to DRβ1-Arg74-positive cells at different doses-10 μM (D), 20 μM (E) or 50 μM (F). Result of a biotinylated-scrambled thyroglobulin peptide scr2098 (negative control) binding to DRβ1-Arg74 -positive cells at 50 μM is shown in (G). APC streptavidin (x-axis) was used to detect binding of the peptide to VAVY cells. L243 and donkey anti-mouse IgG-PE was used to detect DRβ-Arg74 expression (y-axis). Details are described in Materials and Methods. The upper right-hand quadrant indicates the percentage of biotinylated TSHR peptides binding to DRβ1-Arg74positive cells.

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Fig. 4. T-cell proliferation results of NOD-DR3 mice immunized with TSHR.132 (n = 9) (A) or TSHR.197 (n = 8) (B). Stimulation index ≥2 was considered positive. Interferon gamma, IL-2, IL-4 and IL-10 production by NOD-DR3 mice immunized with TSHR.132 (n = 9) (C–F) or TSHR.197 (n = 8) (G–J). NC, negative control peptide. Anti-mouse CD3/CD28 beads, positive control. There are 4 replicates in each experimental group.

Fig. 5. T-cell responses of BALB/c-DR3 mice induced with EAGD. Interferon gamma (A), IL-2 (B), IL-4 (C) and IL-10 (D) production when BALB/c-DR3 mice were immunized with adenovirus expressing the A-subunit of TSHR (n = 19) or the LacZ gene control (n = 16). Statistically significant response to TSHR.132 compared to medium control was observed. In contrast, wild type BALB/c mice did not produce statistically significant amount of interferon gamma (E), IL-2 (F), IL-4 (G) or IL-10 (H) when immunized with the adenovirus expressing TSHR (n = 5). Each experimental group has 4 replicates. *, p < 0.05, ***, p < 0.001. NS, not significant.

serving as a negative control. BALB/c-DR3 mice were immunized with AdTSHR (or AdLacZ control) at week 0, week 3 and week 6, and sacrificed at week 9. Mice immunized with AdLacZ control did not show T-cell responses to any of the peptides, when compared to medium control (Fig. 5A–D, n = 16). Both Th1 and Th2 T-cell responses to TSHR.132 were observed in mice immunized with AdTSHR (n = 19) as indicated by significantly higher levels of interferon gamma (31.6 pg/ ml, p = 0.001) (Fig. 5A), IL-2 (23.5 pg/ml, p < 0.001) (Fig. 5B), IL-4 (6.44 pg/ml, p < 0.001) (Fig. 5C) and IL-10 (7.72 pg/ml, p = 0.0156) (Fig. 5D) compared to medium control. There was no significant increase in interferon gamma, IL-2, IL-4 and IL-10 production in response

to stimulation with TSHR.197 compared to medium control. An unrelated peptide (APO) was used as a negative control. Anti-mouse CD3/ CD28 beads were used as a positive control. In order to confirm that TSHR peptides were being presented within the human HLA-DR3 expressed in the BALB/c-DR3 mice we immunized wild type BALB/c mice (that do not express human DR3) with AdTSHR using the same protocol. Lymphocytes isolated from WT BALB/c mice immunized with AdTSHR did not show increased interferon gamma, IL2, IL-4 and IL-10 cytokine production in response to TSHR.132 and TSHR.197 compared to medium control (Fig. 5E–H, n = 5), indicating that TSHR.132 was eliciting T-cell responses in BALB/c-DR3 mice by 7

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Fig. 6. TSHR antibody (TRAb) production (expressed in fold increase over the positive cut-off value; details described in Materials and Methods) (A), free T4 (B) and total T3 levels (C) in BALB/c-DR3 mice immunized with adenovirus expressing the A-subunit of TSHR or LacZ gene control. TRAb: AdLacZ (n = 31), AdTSHR (n = 34); Free T4: AdLacZ (n = 23), AdTSHR (n = 29); Total T3: AdLacZ (n = 31), AdTSHR (n = 34).**, p < 0.01; ***, p < 0.001.

found to block peptide binding to HLA-DRβ1-Arg74, can block T-cell responses to Tg in the EAT animal model of Hashimoto's thyroiditis induced in NOD-DR3 mice [23]. Therefore, we next tested if Cepharanthine can block a pathogenic TSHR peptide, TSHR.132, from stimulating T-cell responses. We tested the potency to inhibit TSHR.132 binding in our in vitro ELISA by testing the inhibition of TSHR.132 binding to HLA-DRβ1-Arg74 at decreasing Cepharanthine concentrations (0.0125–0.4 mM). Cepharanthine inhibited TSHR.132 binding to recombinant HLA-DRβ1-Arg74 in a dose-dependent manner, with approximate IC50 of 0.125 mM (Fig. 8).

being presented within HLA-DR3. 3.7. TRAb production in BALB/c-DR3 mice immunized with AdTSHR BALB/c-DR3 mice that were immunized with AdTSHR (n = 34) had significantly higher levels of TRAb (13 fold, p < 0.001) when compared to BALB/c-DR3 mice immunized with AdLacZ control (1.09 fold, n = 31) (Fig. 6A). 3.8. Sera of BALB/c-DR3 mice immunized with AdTSHR contain antibodies that stimulate TSHR-dependent cAMP production in CHO-HA-TSHR luciferase cells

3.13. Cepharanthine blocks T-cell responses to TSHR.132 ex vivo in the EAGD model

Sera from BALB/c-DR3 mice immunized with AdTSHR or AdLacZ were tested for the cAMP-stimulating capacity of the TSHR antibodies they contain. Luciferase activity was measured upon incubation of the sera with the CHO-HA-TSHR luciferase cells. Average luciferase activity of 426.3 units (standard deviation = 223.95) was measured in sera from 12 AdLacZ-immunized mice, while significantly higher average luciferase activity of 1238.3 units was measured in the sera from 14 AdTSHR-immunized mice (standard deviation = 1112.08, p = 0.0103) (Supplementary Fig. 1). This indicated that the sera of the AdTSHRimmunized mice contained stimulating TSHR antibodies.

Next we tested if Cepharanthine can block TSHR.132 presentation to T-cells in the BALB/c-DR3 EAGD model. BALB/c-DR3 mice were immunized with AdTSHR and isolated splenocytes were stimulated with TSHR.132 with or without Cepharanthine (S53) (n = 15). DMSO (solvent used to dissolve Cepharanthine) was used as a control. Our results showed that Cepharanthine blocked interferon gamma production by stimulated T-cells (p = 0.0102) (Fig. 9A) and IL-2 production (p = 0.0093) (Fig. 9B) in response to TSHR.132, while DMSO did not. However, Cepharanthine did not block IL-4 production in response to TSHR.132 (Fig. 9C). BALB/c-DR3 mice immunized with AdLacZ (n = 15) did not show an increase in any cytokine production, nor did Cepharanthine influence their production levels (Fig. 9E–H).

3.9. Free T4 production in BALB/c-DR3 mice immunized with AdTSHR BALB/c-DR3 mice immunized with AdTSHR (n = 29) produced a significantly higher concentration of free T4 (18.9 pg/ml, p = 0.0021) compared to the mice immunized with AdLacZ control (14.3 pg/ml, n = 23) (Fig. 6B).

3.14. A molecular basis for TCR activation by TSHR peptides In an attempt to dissect the structural basis for the difference in the stimulatory potencies of TSHR.132 and TSHR.197 we examined the properties of the contact residues that are potentially presented to the T-cell Receptor (TCR) using molecular dynamics simulations. Several structural studies established a consensus for the residues that are presented to the TCR loops; residues in position P2, P5 and P7 showed conservation in their interaction with the TCR [34]. Fig. 10 displays our working model showing the residues that are predicted to be presented towards the TCR interface. The peptides lie in the groove of DR3 and the residues point outside of the groove. The complex of TSHR.132 with DR3 presents to the TCR a Phe in P2, Leu in P5 and Lys in P7. In contrast the DR3•TSHR.197 complex presents Lys in P2, Ala in P5 and Tyr in P7. Clearly, the two molecular signatures are quite different. The aromatic Phe (2) is replaced by a charged Lys, the large aliphatic Leu (5) is replaced by a small Ala and the charged Lys (7) by Tyr. It is clear from Fig. 10 that Tyr (7) in TSHR.197 induces a change in the walls of the groove that prevent it from presenting its side chain to the outside. In contrast, Lys (7) in TSHR.132 is prominently exposed to potential interactions with the loops of TCR. We therefore conclude that the molecular signature for TCR activation is probably more similar to that

3.10. Total T3 production in BALB/c-DR3 mice immunized with AdTSHR BALB/c-DR3 mice immunized with AdTSHR (n = 34) produced a significantly higher concentration of total T3 (1.36 ng/ml, p = 0.0019) compared to the mice immunized with AdLacZ control (0.33 ng/ml, n = 31) (Fig. 6C). 3.11. Histology Compared to the AdLacZ-immunized mice (Fig. 7A and C), thyroids from AdTSHR-immunized mice showed enlarged follicles with proliferation of thyroid epithelial cells that were protruding into the colloid space (Fig. 7B and D). 3.12. Cepharanthine blocks TSHR.132 binding to HLA-DRβ1-Arg74 in vitro We have previously shown that Cepharanthine, a compound that we 8

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Fig. 7. Histological sections of BALB/c-DR3 mice induced with experimental autoimmune Graves' disease (EAGD) and control mice. Thyroid of a control mouse that was immunized with AdLacZ is shown in (A) and (C), at magnification of 5× and 10×, respectively. Shown are normal thyroid follicles with thyroid epithelial cells lining the colloid. Thyroid of a mouse that was immunized with AdTSHR-289 (A-subunit) and developed EAGD is shown in (B) and (D), at magnification of 5× and 10×, respectively. Scale bars are shown on the bottom left of each figure. The follicles are enlarged and the thyroid epithelial cells are proliferating and showing invagination into the colloid space (green arrow in D).

pathogenic peptides of the TSHR reside in its ECD. The ECD comprises a leucine-rich domain (LRD, amino acid residues 22–260) with 10 leucine rich repeats, followed by a hinge region of approximately 130 amino acids [35,37,38]. It is believed that the LRD is the main interacting site for TSH and TSHR autoantibodies and may also be the key antigenic part of the TSHR [39]. Our study used a novel approach to identify the major pathogenic peptide in the TSHR ECD based on their binding affinity to the key GD HLA-DR risk variant, HLA-DRβ1-Arg74 [5,40]. We hypothesized that the presentation of major immunogenic TSHR peptide by HLA-DRβ1-Arg74 to CD4+ T-cells is the key step in triggering GD, as it would trigger TSHR autoreactive CD4+Th-cell responses, leading to activation of B-cells and production of TSHR stimulating antibodies causing thyrotoxicosis and clinical GD. In order to identify the major TSHR pathogenic peptides that are presented within HLA-DRβ1-Arg74 we performed virtual screening of the TSHR ECD as well as in vitro peptide binding testing to identify potential TSHR peptides that can bind to HLA-DRβ1-Arg74 protein. Among the 15 TSHR peptides that were predicted to have high binding affinity to HLA-DRβ1-Arg74 (KD < 500 nM) by the virtual screen, we identified two TSHR peptides that showed high binding affinity to HLADRβ1-Arg74 using our unique ELISA assay - TSHR.132 and TSHR.197, both located within the ECD LRD. While the virtual screen is prone to false positive results the sensitivity and reliability of the predictive power of the NetMHCIIpan 3.1 server is illustrated by the fact that 2 out of 15 peptides with the lowest predicted KD's showed the strongest binding in our in vitro assays (Table 1). Furthermore, we confirmed binding of these two peptides to HLA-DRβ1-Arg74 using a cell-based binding assay. To confirm that the strong binding of TSHR.132 and TSHR.197 to DRβ1-Arg74 was selective to the DRβ1-Arg74 pocket variant we tested in silico the binding of TSHR.132 and TSHR.197 to the DRB1*0317 allele that contains a Gln in position 74 of the beta chain (the most protective HLA-DR pocket allele for GD). Indeed, TSHR.132 and TSHR.197 showed significantly lower affinity to DRB1*0317 than to DRβ1-Arg74 (DRB1*0301) confirming the selectivity of their binding to HLA-DRβ1-Arg74. To determine whether these two peptides can be presented to T-cells and activate them, we immunized NOD-DR3 mice (transgenic for human HLA-DRβ1-Arg74 and null for murine class II MHC) with TSHR.132 and TSHR.197. Intriguingly, even though both peptides

Fig. 8. Dose response curve of Cepharanthine inhibiting TSHR.132 binding to HLA-DRβ1-Arg74. Cepharanthine inhibited binding in a dose-dependent manner, at concentrations ranging from 0.4 to 0.0125 mM. The approximate IC50 is 0.125 mM.

in TSHR.132 than in TSHR.197. Further experiments need to corroborate this heuristic conclusion. 4. Discussion The thyroid-stimulating hormone receptor (TSHR) is a G-proteincoupled receptor with seven transmembrane domains. It consists of 764 amino acid residues (84.5 kDa), comprising the A subunit (amino acids 1–289) which includes the majority of the extracellular domain (ECD), and the B subunit (amino acids 290–764) which includes an extracellular segment, and the trans-membrane, and intra-cellular domains. TSHR has a signal peptide (amino acids 1–21) followed by the ECD with 397 residues (amino acids 22–418, 45.2 kDa) [35]. Since the ECD is exposed to immune cells both on the thyroid cell surface and when it is cleaved and shed into the blood [36], we hypothesized that the key 9

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Fig. 9. Results of Cepharanthine (S53) blocking cytokine production in the EAGD model induced in BALB/c-DR3 mice. Interferon gamma, IL-2, IL-4 and IL-10 production (A–D) when BALB/c-DR3 mice were immunized with adenovirus expressing the A subunit of TSHR and stimulated with TSHR peptides (n = 15). Cepharanthine blocked TSHR.132-stimulated production of interferon gamma and IL-2, while DMSO, the vehicle control did not. Interferon gamma, IL-2, IL-4 and IL10 production (E–H) when BALB/c-DR3 mice were immunized with adenovirus expressing the LacZ gene as a control (n = 15). *, p < 0.05; **, p < 0.01.

bound HLA-DRβ1-Arg74 with high affinity only TSHR.132 could activate T-cells in this humanized mouse model. Moreover, in our EAGD model, AdTSHR-immunized mice developed T-cell responses only to TSHR.132 and not to TSHR.197, confirming that TSHR.132 is a major TSHR pathogenic peptide, at least in our “humanized” mouse model of GD and likely in human GD too. We also tested for anti-TSHR.132 antibodies in sera of both NOD-DR3 immunized with TSHR.132 and BALB/c-DR3 mice immunized with AdTSHR, but there was no production of anti-TSHR.132 peptide antibodies (data not shown). In addition, in our EAGD model, we did not observe an eye phenotype or change in mortality rates. Our findings are consistent with those reported by DeGroot and colleagues that also showed TSHR.132 to be a key pathogenic TSHR peptide [15]. In fact to the best of our knowledge TSHR.132 represents the only TSHR peptide identified by two separate research teams, further supporting it as the key pathogenic TSHR peptide triggering GD.

Since TSHR.132 was discovered in our humanized mouse model of GD it is possible that TSHR pathogenic peptides discovered using this model may only be relevant to the humanized mouse model and not to human GD. However, since the mice carry the human HLA-DR3 on all their antigen presenting cells (APC's) we expect the major TSHR pathogenic peptides to be presented by their APC's. Moreover, previous studies in other autoimmune diseases have shown that major human Tcell epitopes also trigger disease in mouse models [41,42]. In view of the significant epitope spreading that occurs in GD epitope-based therapy in GD would be difficult. Therefore, alternative strategies to treat GD, by blocking HLA-DR3 using small molecules [23,43–47] are more likely to be effective as all epitopes will be blocked from binding to HLA-DR3. Indeed, we identified a small compound (Cepharanthine) that can block TSHR peptide presentation to CD4+ Tcells in our humanized mouse model of GD. Cepharanthine is a plant alkaloid extracted from the plant Stephania Cepharantha Hayata. It has

Fig. 10. Depiction of the relative positions of residues P2, P5 and P7 of the TSHR peptides inside the groove of DRβ1-Arg74. Note their different signatures and in particular the difference between the presentation of Lys (7) on TSHR.132 and the induced fold of the wall to accommodate Tyr (7) of TSHR.197. 10

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been widely used in Japan for treatment of a variety of acute and chronic diseases for over 40 years [48–56]. Previously we reported that Cepharanthine can block the binding of thyroglobulin epitopes to DRβ1-Arg74 and their presentation to autoreactive CD4+ T-cells [23]. The fact the Cepharanthine can block both Tg and TSHR peptides from binding to HLA-DRβ1-Arg74 further supports the notion that it will be effective even after epitope spreading has occurred and other peptides are being presented by HLA-DRβ1-Arg74. Taken together with our previous results in the EAT model of HT, these data suggest that Cepharanthine may be developed into a therapeutic drug for AITD, especially since it has a limited side effect profile in humans [48,54], and we have previously shown that Cepharanthine is safe to administer to mice by injections or PO [23]. Blocking the HLADRβ1-Arg74 pocket by a compound such as Cepharanthine will enable us to use a personalized medicine approach to treating AITD, because only the individuals carrying the risk variant (HLA-DRβ1-Arg74) will be treated. Moreover, such an approach will not cause global immunosuppression because only one HLA class II allele (out of 6 possible alleles - two DR, two DQ, and two DP) will be blocked. Further studies on the EAGD model are needed to show whether Cepharanthine can block the development of EAGD in vivo. Importantly, since we have now developed a humanized, HLA-DR3 expressing, BALB/c mouse model for human GD, we can now use it to screen for other small and/ or large molecules that can block peptide binding to HLA-DRβ1-Arg74. In summary, we identified TSHR.132 as a major pathogenic peptide in GD using a humanized mouse model of GD. TSHR.132 is located within the LRD of the TSHR ectodomain, and binds in vitro to HLADRβ1-Arg74, the key HLA-DR pocket signature that triggers GD. It is likely that TSHR.132 participates in the initiation phase of GD. These findings set the stage for developing targeted HLA-DRβ1-Arg74 pocket blocking therapies as a novel therapeutic approach in GD. Indeed, we show here that Cepharanthine can block TSHR peptide presentation to autoreactive CD4+ T-cells in our humanized DR3 expressing mouse model of GD.

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jaut.2020.102402. References [1] G. Barbesino, Y. Tomer, Clinical review: clinical utility of TSH receptor antibodies, J. Clin. Endocrinol. Metab. 98 (2013) 2247–2255. [2] N.R. Rose, R. Bonita, C.L. Burek, Iodine: an environmental trigger of thyroiditis, Autoimmun. Rev. 1 (2002) 97–103. [3] Y. Tomer, T.F. Davies, Infection, thyroid disease, and autoimmunity, Endocr. Rev. 14 (1993) 107–120. [4] Y. Tomer, T.F. Davies, Searching for the autoimmune thyroid disease susceptibility genes: from gene mapping to gene function, Endocr. Rev. 24 (2003) 694–717. [5] Y. Ban, T.F. Davies, D.A. Greenberg, E.S. Concepcion, R. Osman, T. Oashi, et al., Arginine at position 74 of the HLA-DR beta1 chain is associated with Graves' disease, Genes Immun. 5 (2004) 203–208. [6] L.J. DeGroot, J. Quintans, The causes of autoimmune thyroid disease, Endocr. Rev. 10 (1989) 537–562. [7] F. Menconi, M.C. Monti, D.A. Greenberg, T. Oashi, R. Osman, T.F. Davies, et al., Molecular amino acid signatures in the MHC class II peptide-binding pocket predispose to autoimmune thyroiditis in humans and in mice, Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 14034–14039. [8] J. Couet, S. Sar, A. Jolivet, M.T. Hai, E. Milgrom, M. Misrahi, Shedding of human thyrotropin receptor ectodomain. Involvement of a matrix metalloprotease, J. Biol. Chem. 271 (1996) 4545–4552. [9] C.R. Chen, P. Pichurin, Y. Nagayama, F. Latrofa, B. Rapoport, S.M. McLachlan, The thyrotropin receptor autoantigen in Graves disease is the culprit as well as the victim, J. Clin. Investig. 111 (2003) 1897–1904. [10] S. Sakata, S. Tanaka, K. Okuda, K. Miura, T. Manshouri, M.Z. Atassi, Autoimmune Tcell recognition sites of human thyrotropin receptor in Graves' disease, Mol. Cell. Endocrinol. 92 (1993) 77–82. [11] A. Martin, M. Nakashima, A. Zhou, D. Aronson, A.J. Werner, T.F. Davies, Detection of major T cell epitopes on human thyroid stimulating hormone receptor by overriding immune heterogeneity in patients with Graves' disease, J. Clin. Endocrinol. Metab. 82 (1997) 3361–3366. [12] H. Inaba, D. Pan, Y.H. Shin, W. Martin, G. Buchman, L.J. De Groot, Immune response of mice transgenic for human histocompatibility leukocyte Antigen-DR to human thyrotropin receptor-extracellular domain, Thyroid 19 (2009) 1271–1280. [13] H. Inaba, W. Martin, M. Ardito, A.S. De Groot, L.J. De Groot, The role of glutamic or aspartic acid in position four of the epitope binding motif and thyrotropin receptorextracellular domain epitope selection in Graves' disease, J. Clin. Endocrinol. Metab. 95 (2010) 2909–2916. [14] H. Inaba, L. Moise, W. Martin, A.S. De Groot, J. Desrosiers, R. Tassone, et al., Epitope recognition in HLA-DR3 transgenic mice immunized to TSH-R protein or peptides, Endocrinology 154 (2013) 2234–2243. [15] H. Inaba, W. Martin, A.S. De Groot, S. Qin, L.J. De Groot, Thyrotropin receptor epitopes and their relation to histocompatibility leukocyte antigen-DR molecules in Graves' disease, J. Clin. Endocrinol. Metab. 91 (2006) 2286–2294. [16] P. Pichurin, N. Pham, C.S. David, B. Rapoport, S.M. McLachlan, HLA-DR3 transgenic mice immunized with adenovirus encoding the thyrotropin receptor: T cell epitopes and functional analysis of the CD40 Graves' polymorphism, Thyroid 16 (2006) 1221–1227. [17] Y. Okamoto, T. Yanagawa, M.E. Fisfalen, L.J. DeGroot, Proliferative responses of peripheral blood mononuclear cells from patients with Graves' disease to synthetic peptides epitopes of human thyrotropin receptor, Thyroid 4 (1994) 37–42. [18] M. Soliman, E. Kaplan, T. Yanagawa, Y. Hidaka, M.E. Fisfalen, L.J. DeGroot, T-cells recognize multiple epitopes in the human thyrotropin receptor extracellular domain, J. Clin. Endocrinol. Metab. 80 (1995) 905–914. [19] M. Soliman, E. Kaplan, V. Guimaraes, T. Yanagawa, L.J. DeGroot, T-cell recognition of residue 158-176 in thyrotropin receptor confers risk for development of thyroid autoimmunity in siblings in a family with Graves' disease, Thyroid 6 (1996) 545–551. [20] M.E. Fisfalen, E.M. Palmer, G.A. Van Seventer, K. Soltani, Y. Sawai, E. Kaplan, et al., Thyrotropin-receptor and thyroid peroxidase-specific T cell clones and their cytokine profile in autoimmune thyroid disease, J. Clin. Endocrinol. Metab. 82 (1997) 3655–3663. [21] Y. Sawai, L.J. DeGroot, Binding of human thyrotropin receptor peptides to a Graves' disease-predisposing human leukocyte antigen class II molecule, J. Clin. Endocrinol. Metab. 85 (2000) 1176–1179. [22] H. Vlase, M. Nakashima, P.N. Graves, Y. Tomer, J.C. Morris, T.F. Davies, Defining the major antibody epitopes on the human thyrotropin receptor in immunized mice: evidence for intramolecular epitope spreading, Endocrinology 136 (1995) 4415–4423. [23] C.W. Li, F. Menconi, R. Osman, M. Mezei, E.M. Jacobson, E. Concepcion, et al., Identifying a small molecule blocking antigen presentation in autoimmune thyroiditis, J. Biol. Chem. 291 (2016) 4079–4090. [24] M. Andreatta, E. Karosiene, M. Rasmussen, A. Stryhn, S. Buus, M. Nielsen, Accurate pan-specific prediction of peptide-MHC class II binding affinity with improved binding core identification, Immunogenetics 67 (2015) 641–650. [25] C.W. Li, R. Osman, F. Menconi, E.S. Concepcion, Y. Tomer, Flexible peptide recognition by HLA-DR triggers specific autoimmune T-cell responses in autoimmune thyroiditis and diabetes, J. Autoimmun. 76 (2017) 1–9.

CRediT authorship contribution statement Cheuk Wun Li: Formal analysis, Writing - original draft. Roman Osman: Writing - original draft. Francesca Menconi: Formal analysis. Erlinda Concepcion: Supervision. Yaron Tomer: Conceptualization, Formal analysis, Writing - original draft. Declaration of competing interest Dr. Tomer declares that he is a holder of Patent Application PCT/ US2016/067775 for using small molecules to block HLA-DR3. Dr. Tomer also submitted 2 additional provisional patent disclosures that are not related to the content of this manuscript. Dr. 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 manuscript is not related to that research project. The other authors have nothing to declare. Acknowledgements We thank Dr. Chella David from the Mayo Clinic for generously providing us with the humanized NOD-DR3 mice and C57BL/6-DR3 mice. We thank Drs. Rauf Latif and Terry Davies for generously providing us with the CHO-HA-TSHR luciferase cells. We also thank Hillary Guzik for helping us to scan the thyroid slides, utilizing the 3DHistec Pannoramic 250 Flash II slide scanner (Shared Instrumentation Grant # 1S10OD019961-01) and Andrea Briceno for software training (Analytical Imaging Facility at Albert Einstein College of Medicine, funded by NCI cancer center support grant P30CA013330). This work was supported in part by grants DK067555 and DK073681 from NIDDK (to YT). 11

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