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Detection of Major T Cell Epitopes on Human Thyroid Stimulating Hormone Receptor by Overriding Immune Heterogeneity in Patients with Graves’ Disease¹

Division of Endocrinology and Metabolism, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029-6574

Address all correspondence and requests for reprints to: Dr. Andreas Martin, Department of Medicine, Box 1055, Mount Sinai Medical Center, One Gustave L. Levy Place, New York, New York 10029-6574. E-mail: amartin{at}smtplink.mssm.edu

	Abstract

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To examine the major immunogenic regions of the human TSH receptor (hTSHR), we examined 14 patients with Graves’ disease and 14 healthy control subjects for their peripheral blood T cell proliferative responses to 29 synthetic peptides representing the entire ectodomain of the hTSHR (TSHR-ecd). By combining an analytical approach encompassing the grading of peptide-induced responses and nonparametric testing, we obtained evidence for highly significant differences (P = <0.000001) in the patient group compared with minor differences in the control group (P = 0.045). To account for this difference, we identified four major T cell epitopes (amino acid 247–266, 202–221, 142–161, and 52–71), by multiple comparison analysis, in the patient group. Furthermore, we demonstrated by radiolabeled PCR that the responding T cells were clonally expanding.

These findings demonstrate that despite likely differences in human leukocyte antigen type among patients with Graves’ disease, several distinct hTSHR epitopes elicited significant responses in the immune system of patients with Graves’ disease, and that such patients are most often poorly tolerant to particular epitopes of the TSH receptor ectodomain, The data support the notion of TSHR peptide antigens overriding human immune heterogeneity in patients with Graves’ disease, and raise the possibility of applying analog peptide blockade to suppress T cell responsivity.

	Introduction

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THE HUMAN TSH receptor (hTSHR) is the major autoantigen in Graves’ disease and is the binding site for thyroid-stimulating autoantibodies (TSHR-Ab) (1). The hTSHR consists of a 100-kilodalton, glycosylated 744 amino acid sequence. An extracellular domain (ecd) is encoded by the first 9 exons of the gene and part of the last exon, whereas transmembrane and intracellular regions are encoded only by the 10th exon. Seven hydrophobic transmembrane spanning regions in the hTSHR indicate that it is a member of the G protein-coupled receptor gene superfamily. The TSHR has been found to be proteolyzed to two subunits, and ß, which are linked by disulfide bonds (1, 2, 3, 4). The transmembrane region, with its 3 extracellular loops and 3 cytoplasmic loops, has highly conserved 1st, 3rd, and 7th domains and is, overall, 70–75% homologous with the LH/CG receptor and has regions of great importance in TSHR activation and inactivation (5, 6, 7).

The most nonconserved regions of the ecd of the hTSHR, compared with other members of this G protein-linked supergene family, are amino acid residues 38–45 and 317–366. Although these areas appear to be critical for TSH binding (8, 9), there is considerable tolerance for homologous substitution in all areas of the ecd, suggesting that the TSH binding site involves the entire region (10). Hence, such a large, nonlinear region may be the site of multiple epitopes, and binding to any of them by hTSHR-Ab may disrupt TSH binding to its receptor to varying degrees. Although a solubilized, crude hTSHR preparation was shown in 1978 to induce T cell activation as measured by lymphocyte transformation (11), there have only recently been studies of the hTSHR T cell epitopes in human autoimmune thyroid disease and in animals immunized with hTSHR protein. The availability of recombinant hTSHR-ecd and the full protein sequence made such studies feasible, using both intact receptor and receptor peptides. T cell epitope recognition has been reported to be highly variable in patients with autoimmune thyroid disease and normal individuals have also been shown to react to hTSHR-ecd peptides (12, 13, 14, 15, 16, 17, 18, 19, 20, 21).

As T cells constitute the controlling arm of the immune system, their reactivity to synthetic hTSHR peptides may further our understanding of hTSHR recognition. However, the published reports discussed above lack consensus about the range and pattern of responses to hTSHR peptides. In the present report, we reevaluated such peptide responses using a response grading technique to enable us to assess groups of normal subjects and patients with Graves’ disease, and we identified four T cell epitopes. Such observations may have a profound influence on our understanding of TSHR tolerance and on our ability to control the immune response to the hTSHR.

	Materials and Methods

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Patients and controls

Heparinized blood was obtained from patients with the diagnosis of Graves’ disease as defined by a past history of hyperthyroidism and the presence of hTSHR autoantibodies (22). Normal controls had no history of autoimmune thyroid disease. A total of 14 patients (12 females, 2 males; mean age 49.9 ± 2.8 yr) and 14 controls (9 females, 5 males; mean age 37.7 ± 3.0 yr) were tested. Patients were sequential, regular clinic patients, with clinically established Graves’ disease. Eight patients were euthyroid after treatment with radioactive iodide, and six were recently diagnosed.

hTSHR-ecd peptides

Twenty-nine synthetic peptides (overlapping by 5 amino acids) (kindly supplied by Dr. John Morris, Mayo Clinic, Rochester, MN) were based on the extracellular sequence of the hTSHR and included the three extracellular transmembrane domain loops (EC1, EC2, and EC3) as published previously (23). We have renumbered the peptides to include the signal sequence [21 amino acids (5)], i.e. the first peptide in our analysis begins with residue 22. Peptide lengths varied from 12–21 amino acids and were purified by high performance liquid chromatography. Peptides were checked for purity by amino acid composition analysis and mass spectrometrty; their purity was greater than 95%.

T cell proliferation assays

Peripheral blood mononuclear cells (PBMC) were Ficoll-separated from peripheral blood and incubated in the wells of round-bottom Linbro microtiter plates (2 x 10⁵ cells/well) (Flow Laboratories, McLean, VA) in triplicate with 50 µg/mL peptide for 6 days in medium RPMI 1640 (Gibco, Grand Island, NY) supplemented with 1% penicillin/streptomycin, 25 mM HEPES, and L-glutamine in the presence of 10% heparinized human plasma. Cells were then labeled with tritiated thymidine (0.5 µCi = 18.5 kBq/well) for an additional 18 h, subsequently harvested (PHD cell harvester, model 200A; Cambridge Technology, Cambridge, MA) and subjected to liquid scintillation counting (Beckman Instruments, Fullerton, CA). The median of each triplicate was used for statistical analysis (see below).

RNA extraction and RT

Total cellular RNA was extracted using guanidinium thiocyanate and phenol (RNAzol B, Cinna/Biotecx Labs. Houston, TX) and stored at -70 C in sterile diethylenpyrocarbonate (Sigma, St. Louis, MO)-treated water. Complementary DNA (cDNA) transcripts were prepared from 1 µg cellular RNA using oligo-dT (1 µg/20 µL vol) and avian reverse transcriptase (30 U/20 µL (Life Sciences, St. Petersburg, FL) in the presence of RNAsin (Promega Corp., Madison, WI). The reactions were diluted with 200 µL sterile water and stored at -20 C. Cellular RNA was extracted from PBMC using the same technique and transcribed into cDNA.

Radiolabeled PCR and sequencing

The radiolabeled RT-PCR was based on the differing lengths of the human T cell receptor (TcR) complementarity-determining region 3 (CDR3), which is subject to random nucleotide additions and deletions. Hence, individual T cell clones may have CDR3 regions of different lengths that can be visualized as distinct bands for each human TcR (hTcR) V gene family. hTcR V and Vß constant region oligonucleotides (C: 5'-GGTGAATA GGCAGACAGACTTGTCACTGGA-3', and Cß: 5'-GCCCCTGGCCAAGCACACGAGCGTAGCCTT-3') were labeled with a [³²P]ATP (NEN Research Products, Boston, MA) and T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Eighteen different human V and 21 human Vß gene oligonucleotides were used as forward amplimers, and the ³²P-labeled constant region oligonucleotides were used as reverse amplimers as described previously (24). For the PCR reactions, 5 µL denatured cDNAs were amplified in a 20 µL final volume with 1 U Taq polymerase (Promega), 50 ng of each primer, 200 nM of each deoxynucleotide triphosphate (Boehringer Mannheim, Indianapolis, IN), and Taq polymerase buffer containing 1.5 mM MgCl₂. A 35-cycle step program (95 C for 1 min, 60 C for 2 min, and 72 C for 3 min) was followed by a 10-min extension at 72 C (programmable thermal controller, M.J. Research, Cambridge, MA). Six microliters of the reactions were mixed with 4 µL 95% formamide, 20 mM EDTA, and 0.05% xylene cyanol, and then heated to 95 C for 5 min. Four microliters of the mixture was applied to a 6% sequencing polyacrylamide gel and subjected to electrophoresis at 1700 V for 5.5–6.5 h. Samples were applied in sequence in keeping with their predicted sizes so that all products reached a similar region on the gel at the end of the electrophoresis.

Sequencing of PCR products

Where selected, RT-PCR products were further amplified for 35 cycles (with the same V and Vß primers and an internal C primer) using DNA rescued from the acrylamide gels. Reamplified products were subjected to electrophoresis, and DNA of appropriate predicted size was isolated and purified using Geneclean II (BIO 101, Vista, CA). The purified DNA was then inserted into a TA cloning vector (In Vitrogen Corp., San Diego, CA) and used to transform competent Escherichia coli. Plasmid DNA inserts were prepared from colonies and sequenced by the dideoxy chain termination method using a Sequenase v2.0 kit (United States Biochemical Corp, Cleveland, OH).

Statistical analysis

We used a new approach to the analysis of T cell responses. To give each subject equal weight in our overall group analysis, we first graded the median of triplicate antipeptide responses for each patient. Responses were graded within each patient (experiment), with the highest median response (cpm) receiving the highest number (grade) of 29, and the lowest median response receiving a grade of 1. Subsequently, the grades were analyzed by ANOVA on ranks (Kruskal-Wallis), because the data were not normally distributed. Grades from both groups (patients and controls) were subjected to this nonparametric procedure. The Student-Newman-Keuls test was then used to isolate significantly different antipeptide responses. Results were compared with baseline (lowest median grade) in each group. The grading approach required equal group size, therefore only experiments with a complete set of 29 peptides were used.

	Results

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Identification of peptides giving significant proliferative responses

Four peptides (amino acids 247–266, 202–221, 52–71, and 142–161) elicited significant responses (Fig. 1A). This was in contrast to the normal controls in which only one peptide (amino acid 217–236) was significantly different from the baseline (Fig. 1B). Peptide 187–206 served as baseline in both patients and controls, because it had the lowest stimulating activity.

View larger version (35K): [in this window] [in a new window]   Figure 1. A, Illustration of mean stimulation index of peptide responses in patient group. As baseline we chose the peptide with lowest median grade (peptide 187–206). Four peptide responses differed significantly (nonparametric analysis, see Materials and Methods) and were isolated by Student-Newman-Keuls multiple comparison test (amino acid 247–266, 202–221, 52–71 and 142–161, P < 0.05, indicated by asterisks). Overall P value for interpeptide differences was 0.000000299 in patient group (ANOVA on ranks, Kruskal-Wallis). B, Illustration of mean stimulation index of peptide responses in normal control group. As baseline we again chose (peptide 187–206, identical to patient group). Only one peptide differed significantly (nonparametric analysis) (asterisk, P < 0.05, Student-Newman-Keuls test) and from only two other peptides (those with lowest grades). Overall P value for interpeptide differences was 0.045) in normal control group (ANOVA on ranks, Kruskal-Wallis).

Example of T cell response and dose-response curve

An example of T cell reactivity to all 29 hTSHR-ecd peptides in a patient with Graves’ disease is shown in Fig. 2. A dose-response relationship to stimulating peptide 202–221 (50, 10, 2, and 0.04 µg/mL) derived from another patient is shown in Fig. 3, which also illustrates the specificity of the antipeptide T cell response. Data for individual responses were illustrated as mean values ± SEM.

View larger version (28K): [in this window] [in a new window]   Figure 2. Illustration of T cell reactivity against 29 synthetic peptides of human TSH receptor. Bars represent cpm (mean ± SEM in contrast to Fig. 1, A and B in which stimulation indices were based on median values). Note relatively vigorous response to one particular peptide (142–161); one of four peptides found significant in the overall analysis. This response was further investigated using radiolabeled PCR (see Fig. 4, Table 1, and text).

View larger version (16K): [in this window] [in a new window]   Figure 3. Dose-response curve of patient response to synthetic TSHR peptide 202–221 (one of four significant peptides). Note broad range over which response was maintained (10–50 µg/ml) and decline at lower peptide concentrations. Points represent cpm (mean ± SEM in contrast to Fig. 1, A and B in which stimulation indices were based on median values).

To define whether the response to hTSHR-ecd peptides were either clonal or nonclonal, we used a radiolabeled RT-PCR. PBMC from a patient with Graves’ disease showed a significant proliferative T cell response towards peptide 142–161 (SI = 5.3, t test, P < 0.05, Fig. 4A). cDNAs from uncloned T cells cultured for 6 days with peptide 142–160 or control peptide were examined for their hTcR V gene repertoire. Unstimulated PBMC gave a 6- to 12-band pattern for each of the V gene families examined. However, markedly enhanced bands were observed in certain PCR products from the cultures of peptide 142–161-stimulated T cell cultures (Fig. 4B). These bands were seen in a limited set of V gene families (V14, V15, Vß1, and Vß7). The T cell expansion was further evaluated by sequencing the cloned human TcR CDR3s of Vß1 and Vß7 (8–9 bacterial colonies per V gene family. Eight out of nine sequences for Vß1 (88.9%) and five out of eight (62.5%) sequences for Vß7 showed complete identity of their CDR3 regions and their J segments (Jß1.1 for Vß1, and Jß2.1 for Vß7) indicative of T cell clonal expansion (Table 1). These data indicated, therefore, that hTSHR-ecd peptides initiated T cell proliferation that consisted of both clonal and nonclonal T cell expansion. Similar expanded bands were also found for V14 and V15 (data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 4. A, T cell response to peptide 142–161. Peptide 307–326 served as control. T cells were subsequently analyzed by radiolabeled PCR (see B). Patient had treated Graves’ disease (a 70-yr-old female with pretibial myxedema and a TSHR-antibody titer of 87%) and had twice (in a 5-yr interval) undergone radioiodine therapy before T cell analysis. B, Radiolabeled PCR analysis for 21 Vß families (see text) of T cell response to peptide 142–161 (response is shown in A). For each of 21 Vß gene families, two lanes are shown, one for response to control peptide 307–326 and one to 142–161. Note expanded bands in lanes for Vß1 and Vß7 in response to peptide 142–161. Those bands were subsequently shown to contain clonally expanded T cells (Table 1).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The identification of the four peptide epitopes was made by combining a grading approach with nonparametric statistics. Other studies have addressed the problem of major epitopes in a variety of ways, usually starting with an identification of presumed epitopes within a given patient based on ad hoc criteria. As a result, the reported immunodominant TSHR peptides have varied widely in different studies, and there have been many claims of major epitopes in Graves’ disease (12, 13, 14, 15, 16, 17, 18, 19, 20, 21). An aggravating factor has been the sometimes limited number of peptides (13, 25) or the use of selected T cell reagents with their inherent biases caused by in vitro selection and/or limitations in patient number (18, 20). However, there has not yet been a comprehensive overall statistical analysis. The grading allowed us to compare different patients and different experiments to be evaluated collectively by assigning each subject equal weight. This approach facilitated the combined analysis of a diversity of test results. Interestingly, however, one of the epitopes that emerged from our study (247–266) was similar to a dominant peptide recently identified in a multiplex family by segregation analysis (amino acid 248–263, 21 .

Our study is the first, to our knowledge, to provide an across-the-board analysis of several patients using nonparametric statistics to identify shared epitopes rather than to use criteria that depend on individual patient analysis. The existence of such epitopes is important with regard to the development of epitope-based immunotherapy that might benefit a majority of patients, because peptide analogs have been shown to be effective inhibitors of T cell responses (26, 27). It was remarkable that these epitopes stimulated consistently in an outbred population. It was, therefore, equally surprising to find that three of the same epitopes were also the major epitopes in an inbred strain of mice (balb/c) immunized with mouse TSHR-ecd (M. Kita et al., unpublished observations), further supporting the concept of non-major histocompatibility complex-related T cell epitopes. The fact that certain peptides are predominant despite probable heterogeneity in human leukocyte antigen types illustrates the potential of certain peptides to override (to a degree) known human leukocyte antigen restriction. Similarly, it has been shown that T cells recognizing encephalitogenic determinants of myelin basic protein used similar TcR V and Vß families in the presence of major histocompatibility complex differences in rodents (28) and preferential TcR Vß use in patients with multiple sclerosis (29, 30).

Normal individuals also reacted to one peptide (although much less significantly than patients) as has been previously observed (12). This peptide (217–236) was not one of those prominent in the patient group. Therefore, we speculated that it was most likely a cryptic epitope that is normally not presented to the immune system and, therefore, normal individuals would never be tolerized to it (interestingly, this peptide is located between two of the major peptides recognized in Graves’ disease and actually shares a 5 amino acid overlap with peptide 202–221).

Naturally, the question arose as to how many individual patients reacted to these statistically defined major epitopes. Here again, the grading system was helpful: we observed that >71% of patients had median grades >20 with regard to epitopes 247–266 and 202–221 (the highest grade being 25). These two peptides, therefore, contained the major epitopes in Graves’ disease in our study and it was interesting to note that they were located in close proximity, separated by only two peptides. This raises the question whether their position predisposed them to presentation by the immune system. Epitope 202–221 has previously been identified as a major B cell epitope (31) in Graves’ disease. Both these epitopes are hydrophilic, a property predisposing them to exposure to the immune system (32) and possibly to the TcR (33) and shared with immunodominant regions such as that of proteolipid protein (34).

In conclusion, we identified four peptides eliciting significant proliferative T cell responses in a majority of patients with Graves’ disease. Such peptides may provide a useful basis for designing rational immunotherapies.

	Acknowledgments

 
We thank S. Yeung and E. Concepcion for technical help, and the patients and volunteers who donated their blood. We also thank Drs. M. Valentine, J. Leibowitz, L. Ahmad, and A. Chernov for providing patient and control blood samples.

	Footnotes

 
¹ This work was supported in part by Grants DK-35764 and DK-45011 from NIDDKD (to T.F.D.).

² Supported in part by Grant NAG 9–816 from the National Aeronautics and Space Administration.

³ Theodore and Florence Baumritter Professor of Medicine.

Received April 15, 1997.

Revised June 12, 1997.

Accepted June 20, 1997.

	References

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1. Rees Smith B, McLachlan SM, Furmaniak J. 1988 Autoantibodies to the thyrotropin receptor. Endocr Rev. 9:106–121.[Abstract]
2. Russo D, Nagayama Y, Chazenbalk GD, Wadsworth HL, Rapoport B. 1992 Role of amino acids 261–418 in proteolytic cleavage of the extracellular region of the human thyrotropin receptor. Endocrinology. 130:2135–2138.[Abstract]
3. Loosfelt H, Pichon C, Jolivet A, et al. 1992 Two-subunit structure of the human thyrotropin receptor. Proc Natl Acad Sci USA. 89:3765–3769.[Abstract/Free Full Text]
4. Graves PN, Vlase H, Bobovnikova Y, Davies TF. 1996 Multimeric complex formation by the thyrotropin receptor in solubilized thyroid membranes. Endocrinology. 137:3915–3920.[Abstract]
5. Misrahi M, Loosfelt H, Atger M, Sar S, Guiochon Mantel A, Milgrom E. 1990 Cloning, sequencing and expression of human TSH receptor. Biochem Biophys Res Commun. 166:394–403.[CrossRef][Medline]
6. Parmentier M, Libert F, Maenhaut C, et al. 1989 Molecular cloning of the thyrotropin receptor. Science. 246:1620–1622.[Abstract/Free Full Text]
7. Nagayama Y, Kaufman KD, Seto P, Rapoport B. 1989 Molecular cloning, sequence and functional expression of the cDNA for the human thyrotropin receptor. Biochem Biophys Res Commun. 165:1184–1190[CrossRef][Medline]
8. Nagayama Y, Rapoport B. 1992 The thyrotropin receptor 25 years after its discovery: new insight after its molecular cloning. Mol Endocrinol. 6:145–156.[Abstract]
9. Atassi MZ, Manshouri T, Sakata S. 1991 Localization and synthesis of the hormone-binding regions of the human thyrotropin receptor. Proc Natl Acad Sci USA. 88:3613–3617.[Abstract/Free Full Text]
10. Nagayama Y, Wadsworth HL, Russo D, Chazenbalk GD, Rapoport B. 1991 Binding domains of stimulatory and inhibitory thyrotropin (TSH) receptor autoantibodies determined with chimeric TSH-lutropin/chorionic gonadotropin receptors. J Clin Invest. 88:336–340.
11. Mäkinen T, Wägar G, Apter L, von Willebrand E, Pekonen F. 1978 Evidence that the TSH receptor acts as a mitogenic antigen in Graves’ disease. Nature. 275:314–315.[CrossRef][Medline]
12. Tandon N, Freeman MA, Weetman AP. 1992 T cell responses to synthetic TSH receptor peptides in Graves’ disease. Clin Exp Immunol. 89:468–473.[Medline]
13. Fan JL, Desai RK, Seetharamaiah GS, Dallas JS, Wagle NM, Prabhakar BS. 1993 Heterogeneity in cellular and antibody responses against thyrotropin receptor in patients with Graves’ disease detected using synthetic peptides. J Autoimmun. 6:799–808.[CrossRef][Medline]
14. Sakata S, Tanaka S, Okuda K, Miura K, Manshouri T, Atassi MZ. 1993 Autoimmune T-cell recognition sites of human thyrotropin receptor in Graves’ disease. Mol Cell Endocrinol. 92:77–82.[CrossRef][Medline]
15. Okamoto Y, Yanagawa T, Fisfalen MD, DeGroot LJ. 1994 Proliferative responses of peripheral blood mononuclear cells from patients with Graves’ disease to synthetic peptides epitopes of human thyrotropin receptor. Thyroid. 4:37–42.[Medline]
16. Soliman M, Kaplan E, Yanagawa T, Hidaka Y, Fisfalen ME, DeGroot LJ. 1995 T-cells recognize multiple epitopes in the human thyrotropin receptor extracellular domain. J Clin Endocrinol Metab80 :905–914.
17. Nagy EV, Morris JC, Burch HB, Bhatia S, Salata K, Burman KD. 1995 Thyrotropin receptor T cell epitopes in autoimmune thyroid disease. Clin Immunol Immunopathol. 75:117–124.[CrossRef][Medline]
18. Akamizu T, Ueda Y, Hua L, Okuda J, Mori T. 1995 Establishment and characterization of an antihuman thyrotropin (TSH) receptor-specific CD4+ T cell line from a patient with Graves’ disease: evidence for multiple T cell epitopes on the TSH receptor including the transmembrane domain. Thyroid. 5:259–264.[Medline]
19. Soliman M, Kaplan E, Abdel Latif A, Scherberg N, DeGroot LJ. 1995 Does thyroidectomy, radioactive iodine therapy, or antithyroid drug treatment alter reactivity of patients’ T cells to epitopes of thyrotropin receptor in autoimmune thyroid diseases? J Clin Endocrinol Metab. 80:2312–2321.[Abstract]
20. Kellermann S-A, McCormick DJ, Freeman SL, Morris JC, Conti-Fine BM. 1996 TSH receptor sequences recognized by CD4+ T cells in Graves’ disease patients and healthy controls. J Autoimm. 8:695–698.
21. Soliman M, Kaplan E, Guimaraes V, Yanagawa T, DeGroot LJ. 1996 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. 6:545–551.[Medline]
22. Shewring G, Rees Smith B. 1982 An improved radioreceptor assay for TSH receptor antibodies. Clin Endocrinol (Oxf). 17:409–411.[Medline]
23. Morris JC, Bergert ER, McCormick DJ. 1993 Structure-function studies of the human thyrotropin receptor. Inhibition of binding of labeled thyrotropin (TSH) by synthetic human TSH receptor peptides. J Biol Chem. 268:10900–10905.[Abstract/Free Full Text]
24. Davies TF, Martin A, Concepcion ES, Graves P, Cohen L, Ben Nun A. 1991 Evidence of limited variability of antigen receptors on intrathyroidal T cells in autoimmune thyroid disease. N Engl J Med. 325:238–244.[Abstract]
25. Sakata S, Tanaka S-I, Okuda K, Miura K, Manshouri T, Atassi MZ. 1993 Autoimmune T-cell recognition sites of human thyrotropin receptor in Graves’ disease. Mol Cell Endocrinol. 92:77–82.
26. Elias D, Cohen IR. 1996 The hsp60 peptide p277 arrests the autoimmune diabetes induced by the toxin streptozotocin. Diabetes. 45:1168–1172.[Abstract]
27. Elias D, Cohen IR. 1994 Peptide therapy for diabetes in NOD mice. Lancet. 343:704–706.[CrossRef][Medline]
28. Burns FR, Li XB, Shen N, et al. 1989 Both rat and mouse T cell receptors specific for the encephalitogenic determinant of myelin basic protein use similar V alpha and V beta chain genes even though the major histocompatibility complex and encephalitogenic determinants being recognized are different. J Exp Med. 169:27–39.[Abstract/Free Full Text]
29. Wucherpfennig KW, Ota K, Endo N, et al. 1990 Shared human T cell receptor V beta usage to immunodominant regions of myelin basic protein. Science. 248:1016–1019.[Abstract/Free Full Text]
30. Kotzin BL, Karuturi S, Chou YK, et al. 1991 Preferential T-cell receptor beta-chain variable gene use in myelin basic protein-reactive T-cell clones from patients with multiple sclerosis. Proc Natl Acad Sci USA. 88:9161–9165.[Abstract/Free Full Text]
31. Morris JC, Gibson JL, Haas EJ, Bergert ER, Dallas JS, Prabhakar BS. 1994 Identification of epitopes and affinity purification of thyroid stimulating auto-antibodies using synthetic human TSH receptor peptides. Autoimmunity. 17:287–299.[Medline]
32. Vlase H, Nakashima M, Graves PN, Tomer Y, Morris JC, Davies TF. 1995 Defining the major antibody epitopes on the human thyrotropin receptor in immunized mice: evidence for intramolecular epitope spreading. Endocrinology. 136:4415–4423.[Abstract]
33. Martin A, Matsuoka N, Concepcion ES, Davies TF. 1995 Endogenous antigen presentation by autoantigen-transfected Epstein-Barr virus-lymphoblastoid cells: T cell receptor N-region hydrophobicity relates to thyroid antigen recognition. Autoimmunity. 21:223–230.[Medline]
34. Elliott EA, McFarland HI, Nye SH, et al. 1996 Treatment of experimental encephalomyelitis with a novel chimeric fusion protein of myelin basic protein and proteolipid protein. J Clin Invest. 98:1602–1612.[Medline]

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