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Enhancement of experimental Graves' disease by intranasal administration of a T cell epitope of the thyrotropin receptor
Clinical Immunology (2008) 127, 7–13
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y c l i m
Enhancement of experimental Graves' disease by intranasal administration of a T cell epitope of the thyrotropin receptor Takayasu Arima a,⁎, Naoki Shimojo a , Ken-ichi Yamaguchi a , Minako Tomiita a , Leonard D. Kohn b , Yoichi Kohno a a
Department of Pediatrics, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba 260-8670, Japan b College of Osteopathic Medicine, Ohio University, Athens, OH 45701, USA Received 24 April 2007; accepted with revision 14 November 2007 Available online 29 January 2008
KEYWORDS Graves' disease; Mouse model; Thyrotropin receptor; T cell epitope; Intranasal tolerance
Abstract We previously showed that immunization of mice with murine fibroblasts transfected with the thyrotropin receptor (TSHR) and a murine major histocompatibility complex (MHC) class II molecule induces immune thyroid disease with the humoral and histological features of human Graves' disease in about 20% of mice. In this model, based on the proliferative response of T cells from hyperthyroid mice to a panel of overlapping TSHR peptides, we now demonstrate that TSHR 121–140 peptide contains an immunodominant T cell epitope. Supporting this conclusion, spleen cells from mice immunized with TSHR 121–140 peptide showed a strong proliferative response to fibroblasts transfected with the TSHR and a murine I-Ak molecule, but not either alone. Also, intranasal administration of 100 μg of TSHR 121–140 peptide led to suppressed proliferative response of lymph node cells to the peptide. Interestingly, however, administration of this peptide enhanced, rather than suppressed, the frequency and severity of Graves' disease induced by the immunization of the fibroblasts transfected with the TSHR and a murine I-Ak molecule. Spleen cells from hyperthyroid mice that were pretreated with intranasal peptide tended to produce lesser amounts of IL-4, IL-10 and IFN-gamma than those from normothyroid control mice. Although precise mechanisms of this enhancement remain to be determined, the results suggest that attempts to treat Graves' disease by intranasal administration of an immunodominant TSHR T cell epitope may aggravate, not prevent, the disease. © 2007 Elsevier Inc. All rights reserved.
Introduction
⁎ Corresponding author. Fax: +81 43 226 2145. E-mail address: [email protected] (T. Arima).
Mice immunized with fibroblasts expressing an MHC class II molecule and human thyrotropin receptor (hTSHR), but not either alone, develop major features characteristic of Graves' disease (GD), such as thyroid-stimulating autoantibodies
1521-6616/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2007.11.007
8 directed against TSHR, increased serum thyroid hormone levels, and enlarged thyroid glands [1]. The results indicate the need for the simultaneous expression of a class II molecule and the TSHR on the surface of the fibroblasts to develop stimulating anti-TSHR antibodies and full-blown GD in our model. Our findings support the existence of an important association between aberrant class II molecules and the development of autoimmune thyroid disease, as first hypothesized in the early 1980s [2]. The importance of class II molecules on the cells that express TSHR in this model is consistent with a report by Sospedra et al. who describe the hyperinducibility of class II molecules on thyrocytes from GD patients [3]. MHC class II molecules on thyrocytes can present the TSHR, one of several endogenous thyroid antigens, to T cells [4]. However, so far, there has been no report that identifies T cell epitopes of the TSHR, which are actually presented by class II molecule on the TSHR-expressing cells in hyperthyroid subjects. Mucosal tolerance describes a state of lymphocyte hyporesponsiveness to protein antigens applied across mucosal surfaces by oral or nasal instillation. Inducing specific mucosal immune hyporesponsiveness to T cell epitopes of self antigens associated with cell-mediated and antibody-mediated autoimmune diseases [5–9] offers the basis of an appealing therapy with the potential to replace current nonspecific immunosuppressive drugs that may compromise normal effector functions of lymphocytes involved in immune surveillance and protective immunity. Thus far there have been no reports on the epitope-based immunotherapy of Graves' disease. This is partly due to difficulty in identification of immunodominant TSHR T cell epitopes in Graves' disease. We took advantage of our mouse Graves' disease model to identify a TSHR T cell epitope. In this report, we identified TSHR epitopes by examining the proliferative response of Tcells from hyperthyroid mice to an array of overlapping TSHR peptides. We then tested whether intranasal administration of a peptide containing one such immunodominant TSHR T cell epitope could suppress the development of experimental Graves' disease. Intranasal administration of a peptide containing an immunodominant TSHR epitope peptide suppressed the proliferative response and cytokine production of T cells, however, it unexpectedly enhanced Graves' disease.
Materials and methods
T. Arima et al. flask maintained at 37 °C, in 7% CO2, and containing RPMI1640 supplemented with 10% fetal bovine serum, 2 × 10− 5 M 2-mercaptoethanol plus antibiotics. During this incubation period, spleen cells were co-cultured with 1 × 106 of RT4.15HP-TSHR cells that were pretreated with mitomycin C (Kyowa Hakko Kogyo, Co. Ltd., Tokyo, Japan). After 10 days, the primed responder cells were placed in 24 well culture plates and restimulated with 1 × 106 of mitomycin Ctreated RT4.15HP-TSHR cells in the presence of 20 U/ml rIL-2 (Takeda Chemical Industries, Osaka, Japan) for 7 days. Following this secondary stimulation, responder cells were plated at limiting dilutions in 96 well culture plates, 10 cells per well, together with 3 × 10 4 mitomycin C-treated RT4.15HP-TSHR cells, 20 U/ml rIL-2, and 1 × 106 feeder spleen cells from syngeneic mice. The resultant cell line established by this procedure was CD4 positive by flowcytometry (data not shown). Antigen specificity was tested by measuring the proliferative response of the cell line when exposed to RT4.15HP-TSHR, RT4.15HP-pSG5, and DAP.3-TSHR cells.
Overlapping peptides spanning extracellular domain of the human TSHR A panel of 80 overlapping 20-mer peptides spanning the extracellular domain of the human TSHR sequence was synthesized by use of the multi-pin method (Chiron Mimotopes Pty. Ltd., Clayton, Australia); each peptide was offset by five residues from the preceding peptide. Thus, each peptide overlapped the preceding and following peptide by 15 residues.
Proliferation assay and cytokine production T cell lines were incubated with the mitomycin C-treated RT4.15HP-TSHR cells in 96 well culture plates. After a 72 h incubation, the cultures were pulsed with [3H] thymidine (0.5 μCi/well) for 16 h. Cells were collected with a harvester and [3 H] thymidine incorporation measured by liquid scintillation spectrometry. Two hundred lymph node cells or spleen cells from mice were stimulated with TSHR peptides for 5 days and incorporated radioactivity was measured as described above. IL-4, IFN-gamma, and IL-10 were measured in spleen cell suspensions incubated for 72 h with a peptide containing TSHR T cell epitope by using commercial ELISA kits (Biosource, Carmarillo, CA, USA).
Mice and fibroblasts Seven-week-old female C3H/He (MHC haplotype H-2k) mice were used in these experiments. The murine L cell fibroblasts were previously reported [1]. RT4.15HP-TSHR cells are murine L cell fibroblasts that express I-Ak molecule and hTSHR. RT4.15HP-pSG5 cells are fibroblasts that express I-Ak molecule but not hTSHR. DAP.3-TSHR cells are fibroblasts that express human TSHR but not I-Ak molecules.
Establishment of TSHR-specific T cell line Thirty million spleen cells from the mice immunized with RT4.15HP-TSHR cells were incubated in a 25 cm2 culture
Induction of hyperthyroidism by immunization with fibroblasts Mice were intraperitoneally immunized every 2 weeks with 107 RT4.15HP-TSHR cells, which had been pretreated with mitomycin C as described previously [1]. Two weeks after the 7th immunization, mice were sacrificed and bled. Commercial radioimmunoassay (RIA) kits were used to measure the ability of antibodies in the serum to inhibit [125I] TSH binding (PSR Limited, Cardiff, UK), i.e. TSH-binding inhibitory immunoglobulin (TBII) activity and serum thyroxine (T4) levels (Dai-ichi Radioisotopes, Tokyo, Japan). For the measurement of thyroid-stimulating autoantibodies (TSAb) levels in the serum, we used a commercial kit (Diagnostic
Enhancement of experimental Graves' disease by intranasal administration of a T cell epitope of the TSHR
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Results Recognition of TSHR by a TSHR-specific T cell line A T cell line was established from a hyperthyroid mouse immunized with RT4.15HP-TSHR. It exhibited a proliferative response when incubated with fibroblasts containing the class II molecule and TSHR, RT4.15HP-TSHR, but not with TSHR-positive class II-negative fibroblasts, DAP.3-TSHR, or with TSHR-negative class II-positive fibroblasts, RT4.15HPpSG5 (Fig. 1).
Identification of TSHR T cell epitopes Figure 1 T cell line recognition of TSHR+/class II+ fibroblast cells. Triplicate aliquots of twenty thousand T cells were stimulated with 2 × 104 of RT4.15HP-pSG5 (TSHR−/class II+) fibroblasts (upper histogram), RT4.15HP-TSHR (TSHR+/class II+) fibroblasts (middle), or DAP.3-TSHR (TSHR+/class II−) fibroblasts (lower) for 72 h pulsed with [3H] thymidine, and harvested. Incorporated radioactivity was measured using a beta-scintillation counter. Error bars represent SEM. The TSHR-specific T cell line showed marked proliferation when incubated with TSHR+/ class II+ fibroblasts but not other fibroblasts.
Hybrids, Inc [DHI], Athens, OH). The TSAb activity was measured by the cyclic adenosine monophosphate (cAMP) responses to a Chinese hamster ovary cell line transfected with the human thyrotropin receptor (CHO-TSHR) and a CREluciferase construct, using the procedures detailed in the manufacturer's instructions (DHI). Sera from 12 non-immunized control mice served as the normal control; the positive control was a human Graves' sera standard (DHI). Data are expressed as percentage of the mean control value ± 2 SD of triplicate wells. The cut-off value based on these controls was 129% above the mean. The level of mouse TSH was measured using RIA kits from Amersham Biosciences (Piscataway, NJ). Hyperthyroidism was defined by a confirmed serum T4 value N 6.6 μg/dl based on our previous experiments [10]. All experiments were approved by the Animal Experimentation Committee of Graduate School of Medicine, Chiba University.
Intranasal treatment with TSHR 121–140 peptide Mice were treated intranasally with 100 μg TSHR 121–140 peptide (amino acid residue: NH3-ALKELPLLKFLGIFNTGLKMCOOH) in 20 μl of phosphate buffered saline (PBS) for 3 consecutive days before immunization with either 100 μg TSHR peptide emulsified in complete Freund's adjuvant (CFA) or with RT4.15HP-TSHR cells. In the control group, mice were intranasally sham-treated with 20 μl PBS before immunization with TSHR peptide in CFA or RT4.15HP-TSHR cells.
Statistics Mann–Whitney's U test was used for statistical analysis. P values below 0.05 were considered significant.
Spleen cells from hyperthyroid mice were cultured with 80 overlapping peptides spanning the extracellular domain of the hTSHR, and the proliferative response of the spleen cells was examined. The spleen cells exhibited a proliferative response when exposed to some TSHR peptides derived largely from the extracellular portion of the TSHR. Their highest response was to TSHR peptide #25 corresponding to amino acid residues 121–140 of the TSHR followed by weak
Figure 2 Spleen cell proliferation from hyperthyroid mouse to overlapping TSHR peptides. The panel of 20-mer overlapping TSHR peptides spanning extracellular domain of TSHR was tested for stimulating activity of spleen cells from hyperthyroid mice. 5 μg/ml peptide was incubated for 5 days with 5 × 104 spleen cells from hyperthyroid mice immunized with RT4.15HP-TSHR fibroblasts (A) or from euthyroid mice immunized with RT4.15HPpSG5 fibroblasts (B); incubations were in triplicate in 96 well plates. Two peptides (#7 and #17) were not tested because of insolubility in the medium. Cell proliferation was measured by [3H] thymidine incorporation at 5 days; results are presented as stimulation index. Error bars represent SEM. Spleen cell proliferation to TSHR peptide #25 (TSHR residue 121–140) was significantly increased in a hyperthyroid mouse, whereas spleen cell hyper-responsiveness to TSHR peptide #25 was not seen in a euthyroid mouse. The experiment was performed twice with the same results; representative data from one experiment are shown.
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T. Arima et al. and then subcutaneously immunized with 100 μg TSHR 121−140 peptide in CFA twice at 7-day intervals. Seven days after the last immunization, mice were sacrificed and popliteal lymph node cells (LNC) were tested for proliferative response to the peptide. As shown in Fig. 4, proliferation of LNC to the TSHR 121–140 peptide was significantly lower in the intranasally peptide-administered mice than in the PBS-administered mice (P = 0.039). The proliferative response of LNC to PPD was, however, not significantly different between mice that were given intranasal peptide and control mice (Fig. 4), suggesting that this was a TSHR peptide-specific suppression.
Enhancement of hyperthyroidism in mice that receive intranasal TSHR 121–140 peptide treatment
Figure 3 TSHR 121–140 peptide-specific T cells recognize TSHR+/MHC class II+ fibroblasts. Mice were subcutaneously immunized with 100 μg TSHR 121–140 peptide emulsified in CFA. Seven days after immunization, 2 × 105 spleen cells were stimulated with TSHR 121–140 peptide, RT4.15HP-pSG5 (TSHR−/class II+) fibroblasts, RT4.15HP-TSHR (TSHR+/class II+) fibroblasts or DAP.3TSHR (TSHR+/class II−) fibroblasts in triplicate in 96 well plates. Cell proliferation was measured by [3H] thymidine incorporation at 72 h. Histograms represent cell proliferation expressed as stimulation index. Data were shown as means ± SEM from three individual mice. Spleen cells from TSHR 121–140 peptide immunized mice exhibited a specific proliferation response in the presence of TSHR 121–140 peptide itself and RT4.15HP-TSHR (TSHR+/MHC class II+) fibroblasts. The experiment was performed twice with the same results; representative data from one experiment are shown.
We questioned whether intranasal administration of this immunodominant TSHR peptide might induce immune hyporesponsiveness to TSHR and thus inhibit the development of antibody-mediated autoimmune disease [9]. One hundred micrograms TSHR 121–140 peptide was administered intranasally 3 times into mice followed by intraperitoneal immunization with 107 RT4.15HP-TSHR 7 times every 2 weeks. Two weeks after the final immunization, we observed that serum T4 and TSAb activities were elevated more than normal limits in 6 out of 10 mice that received TSHR 121–140 peptide before immunization (Table 1). In contrast, only 2 out of 9 control mice that received PBS had high serum T4 and TSAb activity. The levels of serum T4 and TSAb activities were significantly higher in
response to TSHR peptide #16 (amino acid residues 76–95) (Fig. 2A). Spleen cells from mice immunized with control fibroblasts did not respond to this peptide (Fig. 2B).
Recognition of fibroblasts by TSHR 121–140 peptide-specific T cells Spleen cells from mice immunized with TSHR 121–140 peptide were studied for their proliferative response to RT4.15HP-TSHR. Spleen cells from mice immunized with TSHR 121–140 peptide proliferated in response to the peptide but not to a control peptide, TSHR peptide 171–190 (amino acid residue: NH3-AFQGLCNETLTLKLYNNGFT-COOH). Quite importantly, these spleen cells proliferated when incubated with RT4.15HP-TSHR, but not with class IInegative DAP.3-TSHR or TSHR-negative RT4.15HP-pSG5 (Fig. 3).
Suppression of peptide-specific proliferative T cell response by intranasally administered TSHR 121–140 peptide Mice were treated intranasally with 100 μg of TSHR 121−140 peptide in 20 μl PBS for three consecutive days
Figure 4 Peptide-specific LNC proliferation was reduced by intranasal treatment of TSHR 121–140 peptide. Mice were nasally pretreated with 100 μg of TSHR 121–140 peptide or PBS before being immunized with 100 μg TSHR 121–140 peptide emulsified in CFA. Seven days after second immunization, mouse LNC were stimulated with TSHR 121–140 peptide (filled bar), unrelated peptide, or PPD (hatched bars) in vitro. Cell proliferation was measured by [3H] thymidine incorporation at 5 days in triplicate. Error bars represent SEM. Proliferation of LNC in response to the peptide, but not to PPD, was significantly lower in mice pretreated with TSHR 121–140 peptide when compared to cells from PBS pretreated mice (P = 0.039).
Enhancement of experimental Graves' disease by intranasal administration of a T cell epitope of the TSHR
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Table 1 Thyroid function and anti-TSHR antibodies in mice intranasally pretreated with TSHR 121–140 peptide compared to control mice intranasally pretreated with PBS
Cytokine production of spleen cells from TSHR 121–140 peptide treated mice
Pretreatment Mouse T4, before no. μg/dl immunization
TSH, TSAb, TBII, % μIU/ml % control
TSHR 121–140 i.n.
b 0.1 b 0.1 0.3 b 0.1 1.5 b 0.1 b 0.1 b 0.1 b 0.1 b 0.1 0.2±0.8 4.9 3.7 4.3 3.8 3.6 5.1 b 0.1 b 0.1 3.9 4.2±0.6 0.004⁎
We evaluated IL-4, IFN-gamma, and IL-10 levels in culture supernatants of spleen cells of TSHR 121–140-pretreated hyperthyroid mice after in vitro stimulation with TSHR 121– 140 peptide. TSHR 121–140-specific cytokine production was lower in TSHR 121–140-pretreated mice than PBS-pretreated mice (Fig. 5). Suppression of IFN-gamma was statistically significant (P = 0.045) (Fig. 5).
Average ± SD PBS i.n.
Average ± SD P value (⁎P b 0.05)
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9
6.2 13.6 4.0 5.9 5.2 6.6 11.6 7.1 10.0 7.2 7.7±3.0 4.7 4.7 3.8 5.3 4.9 2.8 7.2 7.7 1.3 4.7±2.0 0.027⁎
125 339 75 111 119 130 319 188 302 297 201 ± 102 97 105 90 116 107 85 178 194 81 117 ± 41 0.034⁎
71.2 90.8 58.3 67.6 68.1 64.2 83.4 92.7 88.7 89.7 77.5±12.9 73.3 68.5 82.7 78.5 26.9 91.2 85.7 94.5 93.5 77.2±20.9 0.462
C3H/He mice were intranasally pretreated with 100 μg TSHR 121–140 peptide or PBS for three consecutive days before being immunized with fibroblasts expressing TSHR/MHC class II (as described in Materials and methods). After final immunization, serum thyroxin (T4) and TSH levels of these mice were measured by radioimmunoassay. The activities of thyroid-stimulating antibodies (TSAb) in serum were measured using a CHO-TSHRluciferase system and are expressed as percentage of the mean control value of triplicate experiments. Titers of anti-TSHR antibodies were also measured as percent inhibition of TSH binding (TBII). Serum T4 more than 6.6 μg/dl was considered high. Serum TSH was considered low when it was less than 0.3 μIU/ml. TSAb and TBII activities more than mean + 3 SD of those in control (129% and 20%, respectively) were considered high and such activities are shown in bold characters. Statistical comparisons of means and SD of data were performed using Mann–Whitney's U test.
TSHR 121–140 peptide pretreated mice than in control mice (serum T4 was 7.7 ± 3.0 μg/dl vs. 4.7 ± 2.0 μg/dl, respectively [P = 0.027] and TSAb activities were 201 ± 102% vs. 117 ± 41%, respectively [P = 0.034]) (Table 1). Moreover, serum TSH levels were significantly lower in TSHR 121–140 peptide pretreated mice than in control mice (0.2 ± 0.8 μIU/ml vs. 4.2 ± 0.6 μIU/ml, respectively [P = 0.004]). Meanwhile, serum TBII levels were positive in all mice intranasally pretreated with both TSHR 121–140 peptide and PBS, and not significantly different between the two groups (77.5 ± 12.9% vs. 77.2 ± 20.9%, respectively [P = 0.462]) (Table 1). Intranasal administration of an immunodominant TSHR T cell epitope thus enhanced rather than inhibited disease expression.
Discussion Our observation that fibroblasts expressing both a class II molecule and TSHR (RT4.15HP-TSHR), but not fibroblasts expressing either a class II molecule (RT4.15HP-pSG5) or the TSHR (DAP.3-TSHR), can stimulate a TSHR-specific T cell line established from hyperthyroid mice suggests that the class II molecule on the fibroblasts can present endogenously processed TSHR peptide to T cells. To identify TSHR epitopes presented by RT4.15HP-TSHR we tested spleen cells from hyperthyroid mice with overlapping peptides spanning the extracellular domain of the TSHR. TSHR 121– 140 peptide induced the highest proliferative response of spleen cells. Furthermore, TSHR 121–140 peptide-specific T cells proliferated well in response to RT4.15HP-TSHR but not to either RT4.15HP-pSG5 or DAP.3-TSHR, in the absence of the exogenous antigenic peptide. These data indicate that TSHR 121–140 peptide contains a naturally processed TSHR T cell epitope that is presented by class II moleculepositive fibroblasts. This peptide contains an I-Ak binding motif (Glutamic acid at anchor position 1 and Glycine at anchor position 9) [11,12], consistent with the conclusion that this is a T cell epitope in this mouse model of Graves' disease. Although, in general, the class II molecule is mainly involved in the presentation of exogenous antigens, it has been demonstrated that endogenous proteins can be presented to T cells by the class II molecule. This may especially be the case for transmembrane protein antigens since it has been reported that immunoglobulins [13,14], transferrin receptor, hepatitis B surface antigen [15], TPO, and TSHR [16] are bound to class II molecules and presented to T cells [17–19]. In this report, we describe an attempt to prevent experimental Graves' disease by pretreatment with a peptide that contains an immunodominant T cell epitope of the TSHR. It was reported in a collagen-induced arthritis model that low dose of intranasal administration of the immunodominant peptide of type II collagen might prime T cells, induce cytokines, and enhance the disease. Disease activity was correlated with the level of antigen-specific T cell activation [20]. In the present study the amount of peptide administered intranasally was 100 μg/dose, similar to that in reports showing suppression of the T cell response by intranasal administration of other peptides [5,21]. The finding that TSHR 121–140-specific T cell proliferation was reduced compared to control mice indicates that peptidespecific immune suppression was achieved in our experiments. This is also evidenced by the tendency toward suppression of the 3 cytokines measured. However, despite the down-regulation of the T cell response to this
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Figure 5 Cytokine production of spleen cells stimulated with TSHR 121–140 peptide. IL-4 (A), IFN-gamma (B), and IL-10 (C) were assayed by ELISA in triplicate cell culture supernatants of spleen cells from three mice, each pretreated with TSHR 121–140 peptide or with PBS before intraperitoneal RT4.15HP-TSHR (TSHR+/class II+) fibroblast immunization. Mice were sacrificed 14 days after the last immunization, and spleen cells were challenged in vitro with TSHR 121–140 peptide. Data were presented as mean ± SEM. Intranasal pretreatment with TSHR 121–140 peptide caused a reduction of all the cytokines; the IFN-gamma reduction was statistically significant (P = 0.045).
immunodominant TSHR epitope, Graves' disease was distinctly enhanced both in frequency and severity by intranasal administration of the TSHR epitope peptide rather than prevented. Experimental autoimmune thyroiditis in murine models has been effectively suppressed by oral administration of either porcine [22] or human thyroglobulin [23]. In the former reports, type 2 CD8 T cells which produce IL-4 and TGF-beta mediated the suppression. In the latter experiments, both Th1 and Th2 responses were down-regulated. These data support the hypothesis that thyroglobulininduced thyroiditis is mediated by Th1 cells. In contrast, a Th2 response plays an important role in the development of hyperthyroidism in our mouse model of Graves' disease since it has been shown that alum and pertussis toxin enhanced disease expression, whereas complete Freund's adjuvant (CFA) delayed the disease [24]. Thus, the shift of Th1/2 balance to Th2 by intranasal administration of the TSHR peptide might be a cause of the increased frequency of disease expression and the enhancement of the serum T4 levels in TSHR 121–140 treated mice. However, in our experiments production of murine Th2 cytokines IL-4 and IL-10 by immune cells was not enhanced but rather suppressed by the intranasal administration of the TSHR peptide. Although suppression of IFN-gamma was statistically significant, the ratio of IL-4/ IFN-gamma or IL-10/IFN-gamma was not different between hyperthyroid and normothyroid mice (data not shown). Thus the mechanism of enhancement of hyperthyroidism by intranasal administration of an immunodominant epitope of TSHR in the present study remains to be determined. In conclusion, our present data suggest that mucosal administration of a peptide containing an immunodominant TSHR T cell epitope may aggravate or promote experimental Graves' disease rather than suppress its onset. Although the precise mechanism of enhancement of the disease remains to be determined, this should be taken into consideration when peptide-based immunotherapy for human Graves' disease is contemplated.
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Enhancement of experimental Graves' disease by intranasal administration of a T cell epitope of the TSHR [10] K. Yamaguchi, N. Shimojo, S. Kikuoka, A. Hoshioka, A. Hirai, K. Tahara, L.D. Kohn, Y. Kohno, H. Niimi, Genetic control of antithyrotropin receptor antibody generation in H-2K mice immunized with thyrotropin receptor-transfected fibroblasts, J. Clin. Endocrinol. Metab. 82 (1997) 4266–4269. [11] D.H. Fremont, D. Monnaie, C.A. Nelson, W.A. Hendrickson, E.R. Unanue, Crystal structure of I-Ak in complex with a dominant epitope of lysozyme, Immunity 8 (1998) 305–317. [12] C.A. Nelson, N.J. Viner, S.P. Young, S.J. Petzold, E.R. Unanue, A negatively charged anchor residue promotes high affinity binding to the MHC class II molecule I-Ak, J. Immunol. 157 (1996) 755–762. [13] R.M. Chicz, R.G. Urban, J.C. Gorga, D.A. Vignali, W.S. Lane, J.L. Strominger, Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles, J. Exp. Med. 178 (1993) 27–47. [14] P.A. Reid, C. Watts, Cycling of cell-surface MHC glycoproteins through primaquine-sensitive intracellular compartments, Nature 346 (1990) 655–657. [15] Y. Jin, W.K. Shih, I. Berkower, Human Tcell response to the surface antigen of hepatitis B virus (HBsAg). Endosomal and nonendosomal processing pathways are accessible to both endogenous and exogenous antigen, J. Exp. Med. 168 (1988) 293–306. [16] R.J. Mullins, Y. Chernajovsky, C. Dayan, M. Londei, M. Feldman, Transfection of thyroid autoantigens into EBVtransformed B cell lines. Recognition by Graves' disease thyroid T cells, J. Immunol. 152 (1994) 5572–5580. [17] S. Weiss, B. Bogen, B-lymphoma cells process and present their endogenous immunoglobulin to major histocompatibility complex-restricted T cells, Proc. Natl. Acad. Sci. U. S. A. 86 (1989) 282–286.
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[18] V.L. Yurin, A.Y. Rudensky, S.M. Mazel, J.M. Blechman, Immunoglobulin-specific T–B cell interaction. II. T cell clones recognize the processed form of B cells' own surface immunoglobulin in the context of the major histocompatibility complex class II molecule, Eur. J. Immunol. 19 (1989) 1685–1691. [19] M.S. Malnati, M. Marti, T. LaVaute, D. Jaraquemada, W. Biddison, R. DeMars, E.O. Long, Processing pathways for presentation of cytosolic antigen to MHC class II-restricted T cells, Nature 357 (1992) 702–704. [20] N.A. Staines, C.J. Derry, L. Marinova-Mutafchieva, N. Ali, D.H. Davies, J.J. Murphy, Constraints on the efficacy of mucosal tolerance in treatment of human and animal arthritic diseases, Ann. N.Y. Acad. Sci. 1029 (2004) 250–259. [21] K. Hufnagl, B. Winkler, M. Focke, R. Valenta, O. Scheiner, H. Renz, U. Wiedermann, Intranasal tolerance induction with polypeptides derived from 3 noncross-reactive major aeroallergens prevents allergic polysensitization in mice, J. Allergy Clin. Immunol. 116 (2005) 370–376. [22] V.C. Guimaraes, J. Quintans, M.-E. Fisfalen, F.H. Straus, K. Wilhelm, G.A. Madeiros-Neto, L.J. DeGroot, Suppression of development of experimental autoimmune thyroiditis by oral administration of thyroglobulin, Endocrinology 136 (1995) s 3353–s 3359. [23] K.E. Peterson, H. Braley-Mullen, Suppression of murine experimental autoimmune thyroiditis by oral administration of porcine thyroglobulin, Cell. Immunol. 166 (1995) 123–130. [24] M. Kita, L. Ahmad, R.C. Marians, H. Vlase, P. Unger, P.N. Graves, T.F. Davies, Regulation and transfer of a murine model of thyrotropin receptor antibody mediated Graves' disease, Endocrinology 140 (1999) 1392–1398.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y c l i m
Enhancement of experimental Graves' disease by intranasal administration of a T cell epitope of the thyrotropin receptor Takayasu Arima a,⁎, Naoki Shimojo a , Ken-ichi Yamaguchi a , Minako Tomiita a , Leonard D. Kohn b , Yoichi Kohno a a
Department of Pediatrics, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba 260-8670, Japan b College of Osteopathic Medicine, Ohio University, Athens, OH 45701, USA Received 24 April 2007; accepted with revision 14 November 2007 Available online 29 January 2008
KEYWORDS Graves' disease; Mouse model; Thyrotropin receptor; T cell epitope; Intranasal tolerance
Abstract We previously showed that immunization of mice with murine fibroblasts transfected with the thyrotropin receptor (TSHR) and a murine major histocompatibility complex (MHC) class II molecule induces immune thyroid disease with the humoral and histological features of human Graves' disease in about 20% of mice. In this model, based on the proliferative response of T cells from hyperthyroid mice to a panel of overlapping TSHR peptides, we now demonstrate that TSHR 121–140 peptide contains an immunodominant T cell epitope. Supporting this conclusion, spleen cells from mice immunized with TSHR 121–140 peptide showed a strong proliferative response to fibroblasts transfected with the TSHR and a murine I-Ak molecule, but not either alone. Also, intranasal administration of 100 μg of TSHR 121–140 peptide led to suppressed proliferative response of lymph node cells to the peptide. Interestingly, however, administration of this peptide enhanced, rather than suppressed, the frequency and severity of Graves' disease induced by the immunization of the fibroblasts transfected with the TSHR and a murine I-Ak molecule. Spleen cells from hyperthyroid mice that were pretreated with intranasal peptide tended to produce lesser amounts of IL-4, IL-10 and IFN-gamma than those from normothyroid control mice. Although precise mechanisms of this enhancement remain to be determined, the results suggest that attempts to treat Graves' disease by intranasal administration of an immunodominant TSHR T cell epitope may aggravate, not prevent, the disease. © 2007 Elsevier Inc. All rights reserved.
Introduction
⁎ Corresponding author. Fax: +81 43 226 2145. E-mail address: [email protected] (T. Arima).
Mice immunized with fibroblasts expressing an MHC class II molecule and human thyrotropin receptor (hTSHR), but not either alone, develop major features characteristic of Graves' disease (GD), such as thyroid-stimulating autoantibodies
1521-6616/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2007.11.007
8 directed against TSHR, increased serum thyroid hormone levels, and enlarged thyroid glands [1]. The results indicate the need for the simultaneous expression of a class II molecule and the TSHR on the surface of the fibroblasts to develop stimulating anti-TSHR antibodies and full-blown GD in our model. Our findings support the existence of an important association between aberrant class II molecules and the development of autoimmune thyroid disease, as first hypothesized in the early 1980s [2]. The importance of class II molecules on the cells that express TSHR in this model is consistent with a report by Sospedra et al. who describe the hyperinducibility of class II molecules on thyrocytes from GD patients [3]. MHC class II molecules on thyrocytes can present the TSHR, one of several endogenous thyroid antigens, to T cells [4]. However, so far, there has been no report that identifies T cell epitopes of the TSHR, which are actually presented by class II molecule on the TSHR-expressing cells in hyperthyroid subjects. Mucosal tolerance describes a state of lymphocyte hyporesponsiveness to protein antigens applied across mucosal surfaces by oral or nasal instillation. Inducing specific mucosal immune hyporesponsiveness to T cell epitopes of self antigens associated with cell-mediated and antibody-mediated autoimmune diseases [5–9] offers the basis of an appealing therapy with the potential to replace current nonspecific immunosuppressive drugs that may compromise normal effector functions of lymphocytes involved in immune surveillance and protective immunity. Thus far there have been no reports on the epitope-based immunotherapy of Graves' disease. This is partly due to difficulty in identification of immunodominant TSHR T cell epitopes in Graves' disease. We took advantage of our mouse Graves' disease model to identify a TSHR T cell epitope. In this report, we identified TSHR epitopes by examining the proliferative response of Tcells from hyperthyroid mice to an array of overlapping TSHR peptides. We then tested whether intranasal administration of a peptide containing one such immunodominant TSHR T cell epitope could suppress the development of experimental Graves' disease. Intranasal administration of a peptide containing an immunodominant TSHR epitope peptide suppressed the proliferative response and cytokine production of T cells, however, it unexpectedly enhanced Graves' disease.
Materials and methods
T. Arima et al. flask maintained at 37 °C, in 7% CO2, and containing RPMI1640 supplemented with 10% fetal bovine serum, 2 × 10− 5 M 2-mercaptoethanol plus antibiotics. During this incubation period, spleen cells were co-cultured with 1 × 106 of RT4.15HP-TSHR cells that were pretreated with mitomycin C (Kyowa Hakko Kogyo, Co. Ltd., Tokyo, Japan). After 10 days, the primed responder cells were placed in 24 well culture plates and restimulated with 1 × 106 of mitomycin Ctreated RT4.15HP-TSHR cells in the presence of 20 U/ml rIL-2 (Takeda Chemical Industries, Osaka, Japan) for 7 days. Following this secondary stimulation, responder cells were plated at limiting dilutions in 96 well culture plates, 10 cells per well, together with 3 × 10 4 mitomycin C-treated RT4.15HP-TSHR cells, 20 U/ml rIL-2, and 1 × 106 feeder spleen cells from syngeneic mice. The resultant cell line established by this procedure was CD4 positive by flowcytometry (data not shown). Antigen specificity was tested by measuring the proliferative response of the cell line when exposed to RT4.15HP-TSHR, RT4.15HP-pSG5, and DAP.3-TSHR cells.
Overlapping peptides spanning extracellular domain of the human TSHR A panel of 80 overlapping 20-mer peptides spanning the extracellular domain of the human TSHR sequence was synthesized by use of the multi-pin method (Chiron Mimotopes Pty. Ltd., Clayton, Australia); each peptide was offset by five residues from the preceding peptide. Thus, each peptide overlapped the preceding and following peptide by 15 residues.
Proliferation assay and cytokine production T cell lines were incubated with the mitomycin C-treated RT4.15HP-TSHR cells in 96 well culture plates. After a 72 h incubation, the cultures were pulsed with [3H] thymidine (0.5 μCi/well) for 16 h. Cells were collected with a harvester and [3 H] thymidine incorporation measured by liquid scintillation spectrometry. Two hundred lymph node cells or spleen cells from mice were stimulated with TSHR peptides for 5 days and incorporated radioactivity was measured as described above. IL-4, IFN-gamma, and IL-10 were measured in spleen cell suspensions incubated for 72 h with a peptide containing TSHR T cell epitope by using commercial ELISA kits (Biosource, Carmarillo, CA, USA).
Mice and fibroblasts Seven-week-old female C3H/He (MHC haplotype H-2k) mice were used in these experiments. The murine L cell fibroblasts were previously reported [1]. RT4.15HP-TSHR cells are murine L cell fibroblasts that express I-Ak molecule and hTSHR. RT4.15HP-pSG5 cells are fibroblasts that express I-Ak molecule but not hTSHR. DAP.3-TSHR cells are fibroblasts that express human TSHR but not I-Ak molecules.
Establishment of TSHR-specific T cell line Thirty million spleen cells from the mice immunized with RT4.15HP-TSHR cells were incubated in a 25 cm2 culture
Induction of hyperthyroidism by immunization with fibroblasts Mice were intraperitoneally immunized every 2 weeks with 107 RT4.15HP-TSHR cells, which had been pretreated with mitomycin C as described previously [1]. Two weeks after the 7th immunization, mice were sacrificed and bled. Commercial radioimmunoassay (RIA) kits were used to measure the ability of antibodies in the serum to inhibit [125I] TSH binding (PSR Limited, Cardiff, UK), i.e. TSH-binding inhibitory immunoglobulin (TBII) activity and serum thyroxine (T4) levels (Dai-ichi Radioisotopes, Tokyo, Japan). For the measurement of thyroid-stimulating autoantibodies (TSAb) levels in the serum, we used a commercial kit (Diagnostic
Enhancement of experimental Graves' disease by intranasal administration of a T cell epitope of the TSHR
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Results Recognition of TSHR by a TSHR-specific T cell line A T cell line was established from a hyperthyroid mouse immunized with RT4.15HP-TSHR. It exhibited a proliferative response when incubated with fibroblasts containing the class II molecule and TSHR, RT4.15HP-TSHR, but not with TSHR-positive class II-negative fibroblasts, DAP.3-TSHR, or with TSHR-negative class II-positive fibroblasts, RT4.15HPpSG5 (Fig. 1).
Identification of TSHR T cell epitopes Figure 1 T cell line recognition of TSHR+/class II+ fibroblast cells. Triplicate aliquots of twenty thousand T cells were stimulated with 2 × 104 of RT4.15HP-pSG5 (TSHR−/class II+) fibroblasts (upper histogram), RT4.15HP-TSHR (TSHR+/class II+) fibroblasts (middle), or DAP.3-TSHR (TSHR+/class II−) fibroblasts (lower) for 72 h pulsed with [3H] thymidine, and harvested. Incorporated radioactivity was measured using a beta-scintillation counter. Error bars represent SEM. The TSHR-specific T cell line showed marked proliferation when incubated with TSHR+/ class II+ fibroblasts but not other fibroblasts.
Hybrids, Inc [DHI], Athens, OH). The TSAb activity was measured by the cyclic adenosine monophosphate (cAMP) responses to a Chinese hamster ovary cell line transfected with the human thyrotropin receptor (CHO-TSHR) and a CREluciferase construct, using the procedures detailed in the manufacturer's instructions (DHI). Sera from 12 non-immunized control mice served as the normal control; the positive control was a human Graves' sera standard (DHI). Data are expressed as percentage of the mean control value ± 2 SD of triplicate wells. The cut-off value based on these controls was 129% above the mean. The level of mouse TSH was measured using RIA kits from Amersham Biosciences (Piscataway, NJ). Hyperthyroidism was defined by a confirmed serum T4 value N 6.6 μg/dl based on our previous experiments [10]. All experiments were approved by the Animal Experimentation Committee of Graduate School of Medicine, Chiba University.
Intranasal treatment with TSHR 121–140 peptide Mice were treated intranasally with 100 μg TSHR 121–140 peptide (amino acid residue: NH3-ALKELPLLKFLGIFNTGLKMCOOH) in 20 μl of phosphate buffered saline (PBS) for 3 consecutive days before immunization with either 100 μg TSHR peptide emulsified in complete Freund's adjuvant (CFA) or with RT4.15HP-TSHR cells. In the control group, mice were intranasally sham-treated with 20 μl PBS before immunization with TSHR peptide in CFA or RT4.15HP-TSHR cells.
Statistics Mann–Whitney's U test was used for statistical analysis. P values below 0.05 were considered significant.
Spleen cells from hyperthyroid mice were cultured with 80 overlapping peptides spanning the extracellular domain of the hTSHR, and the proliferative response of the spleen cells was examined. The spleen cells exhibited a proliferative response when exposed to some TSHR peptides derived largely from the extracellular portion of the TSHR. Their highest response was to TSHR peptide #25 corresponding to amino acid residues 121–140 of the TSHR followed by weak
Figure 2 Spleen cell proliferation from hyperthyroid mouse to overlapping TSHR peptides. The panel of 20-mer overlapping TSHR peptides spanning extracellular domain of TSHR was tested for stimulating activity of spleen cells from hyperthyroid mice. 5 μg/ml peptide was incubated for 5 days with 5 × 104 spleen cells from hyperthyroid mice immunized with RT4.15HP-TSHR fibroblasts (A) or from euthyroid mice immunized with RT4.15HPpSG5 fibroblasts (B); incubations were in triplicate in 96 well plates. Two peptides (#7 and #17) were not tested because of insolubility in the medium. Cell proliferation was measured by [3H] thymidine incorporation at 5 days; results are presented as stimulation index. Error bars represent SEM. Spleen cell proliferation to TSHR peptide #25 (TSHR residue 121–140) was significantly increased in a hyperthyroid mouse, whereas spleen cell hyper-responsiveness to TSHR peptide #25 was not seen in a euthyroid mouse. The experiment was performed twice with the same results; representative data from one experiment are shown.
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T. Arima et al. and then subcutaneously immunized with 100 μg TSHR 121−140 peptide in CFA twice at 7-day intervals. Seven days after the last immunization, mice were sacrificed and popliteal lymph node cells (LNC) were tested for proliferative response to the peptide. As shown in Fig. 4, proliferation of LNC to the TSHR 121–140 peptide was significantly lower in the intranasally peptide-administered mice than in the PBS-administered mice (P = 0.039). The proliferative response of LNC to PPD was, however, not significantly different between mice that were given intranasal peptide and control mice (Fig. 4), suggesting that this was a TSHR peptide-specific suppression.
Enhancement of hyperthyroidism in mice that receive intranasal TSHR 121–140 peptide treatment
Figure 3 TSHR 121–140 peptide-specific T cells recognize TSHR+/MHC class II+ fibroblasts. Mice were subcutaneously immunized with 100 μg TSHR 121–140 peptide emulsified in CFA. Seven days after immunization, 2 × 105 spleen cells were stimulated with TSHR 121–140 peptide, RT4.15HP-pSG5 (TSHR−/class II+) fibroblasts, RT4.15HP-TSHR (TSHR+/class II+) fibroblasts or DAP.3TSHR (TSHR+/class II−) fibroblasts in triplicate in 96 well plates. Cell proliferation was measured by [3H] thymidine incorporation at 72 h. Histograms represent cell proliferation expressed as stimulation index. Data were shown as means ± SEM from three individual mice. Spleen cells from TSHR 121–140 peptide immunized mice exhibited a specific proliferation response in the presence of TSHR 121–140 peptide itself and RT4.15HP-TSHR (TSHR+/MHC class II+) fibroblasts. The experiment was performed twice with the same results; representative data from one experiment are shown.
We questioned whether intranasal administration of this immunodominant TSHR peptide might induce immune hyporesponsiveness to TSHR and thus inhibit the development of antibody-mediated autoimmune disease [9]. One hundred micrograms TSHR 121–140 peptide was administered intranasally 3 times into mice followed by intraperitoneal immunization with 107 RT4.15HP-TSHR 7 times every 2 weeks. Two weeks after the final immunization, we observed that serum T4 and TSAb activities were elevated more than normal limits in 6 out of 10 mice that received TSHR 121–140 peptide before immunization (Table 1). In contrast, only 2 out of 9 control mice that received PBS had high serum T4 and TSAb activity. The levels of serum T4 and TSAb activities were significantly higher in
response to TSHR peptide #16 (amino acid residues 76–95) (Fig. 2A). Spleen cells from mice immunized with control fibroblasts did not respond to this peptide (Fig. 2B).
Recognition of fibroblasts by TSHR 121–140 peptide-specific T cells Spleen cells from mice immunized with TSHR 121–140 peptide were studied for their proliferative response to RT4.15HP-TSHR. Spleen cells from mice immunized with TSHR 121–140 peptide proliferated in response to the peptide but not to a control peptide, TSHR peptide 171–190 (amino acid residue: NH3-AFQGLCNETLTLKLYNNGFT-COOH). Quite importantly, these spleen cells proliferated when incubated with RT4.15HP-TSHR, but not with class IInegative DAP.3-TSHR or TSHR-negative RT4.15HP-pSG5 (Fig. 3).
Suppression of peptide-specific proliferative T cell response by intranasally administered TSHR 121–140 peptide Mice were treated intranasally with 100 μg of TSHR 121−140 peptide in 20 μl PBS for three consecutive days
Figure 4 Peptide-specific LNC proliferation was reduced by intranasal treatment of TSHR 121–140 peptide. Mice were nasally pretreated with 100 μg of TSHR 121–140 peptide or PBS before being immunized with 100 μg TSHR 121–140 peptide emulsified in CFA. Seven days after second immunization, mouse LNC were stimulated with TSHR 121–140 peptide (filled bar), unrelated peptide, or PPD (hatched bars) in vitro. Cell proliferation was measured by [3H] thymidine incorporation at 5 days in triplicate. Error bars represent SEM. Proliferation of LNC in response to the peptide, but not to PPD, was significantly lower in mice pretreated with TSHR 121–140 peptide when compared to cells from PBS pretreated mice (P = 0.039).
Enhancement of experimental Graves' disease by intranasal administration of a T cell epitope of the TSHR
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Table 1 Thyroid function and anti-TSHR antibodies in mice intranasally pretreated with TSHR 121–140 peptide compared to control mice intranasally pretreated with PBS
Cytokine production of spleen cells from TSHR 121–140 peptide treated mice
Pretreatment Mouse T4, before no. μg/dl immunization
TSH, TSAb, TBII, % μIU/ml % control
TSHR 121–140 i.n.
b 0.1 b 0.1 0.3 b 0.1 1.5 b 0.1 b 0.1 b 0.1 b 0.1 b 0.1 0.2±0.8 4.9 3.7 4.3 3.8 3.6 5.1 b 0.1 b 0.1 3.9 4.2±0.6 0.004⁎
We evaluated IL-4, IFN-gamma, and IL-10 levels in culture supernatants of spleen cells of TSHR 121–140-pretreated hyperthyroid mice after in vitro stimulation with TSHR 121– 140 peptide. TSHR 121–140-specific cytokine production was lower in TSHR 121–140-pretreated mice than PBS-pretreated mice (Fig. 5). Suppression of IFN-gamma was statistically significant (P = 0.045) (Fig. 5).
Average ± SD PBS i.n.
Average ± SD P value (⁎P b 0.05)
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9
6.2 13.6 4.0 5.9 5.2 6.6 11.6 7.1 10.0 7.2 7.7±3.0 4.7 4.7 3.8 5.3 4.9 2.8 7.2 7.7 1.3 4.7±2.0 0.027⁎
125 339 75 111 119 130 319 188 302 297 201 ± 102 97 105 90 116 107 85 178 194 81 117 ± 41 0.034⁎
71.2 90.8 58.3 67.6 68.1 64.2 83.4 92.7 88.7 89.7 77.5±12.9 73.3 68.5 82.7 78.5 26.9 91.2 85.7 94.5 93.5 77.2±20.9 0.462
C3H/He mice were intranasally pretreated with 100 μg TSHR 121–140 peptide or PBS for three consecutive days before being immunized with fibroblasts expressing TSHR/MHC class II (as described in Materials and methods). After final immunization, serum thyroxin (T4) and TSH levels of these mice were measured by radioimmunoassay. The activities of thyroid-stimulating antibodies (TSAb) in serum were measured using a CHO-TSHRluciferase system and are expressed as percentage of the mean control value of triplicate experiments. Titers of anti-TSHR antibodies were also measured as percent inhibition of TSH binding (TBII). Serum T4 more than 6.6 μg/dl was considered high. Serum TSH was considered low when it was less than 0.3 μIU/ml. TSAb and TBII activities more than mean + 3 SD of those in control (129% and 20%, respectively) were considered high and such activities are shown in bold characters. Statistical comparisons of means and SD of data were performed using Mann–Whitney's U test.
TSHR 121–140 peptide pretreated mice than in control mice (serum T4 was 7.7 ± 3.0 μg/dl vs. 4.7 ± 2.0 μg/dl, respectively [P = 0.027] and TSAb activities were 201 ± 102% vs. 117 ± 41%, respectively [P = 0.034]) (Table 1). Moreover, serum TSH levels were significantly lower in TSHR 121–140 peptide pretreated mice than in control mice (0.2 ± 0.8 μIU/ml vs. 4.2 ± 0.6 μIU/ml, respectively [P = 0.004]). Meanwhile, serum TBII levels were positive in all mice intranasally pretreated with both TSHR 121–140 peptide and PBS, and not significantly different between the two groups (77.5 ± 12.9% vs. 77.2 ± 20.9%, respectively [P = 0.462]) (Table 1). Intranasal administration of an immunodominant TSHR T cell epitope thus enhanced rather than inhibited disease expression.
Discussion Our observation that fibroblasts expressing both a class II molecule and TSHR (RT4.15HP-TSHR), but not fibroblasts expressing either a class II molecule (RT4.15HP-pSG5) or the TSHR (DAP.3-TSHR), can stimulate a TSHR-specific T cell line established from hyperthyroid mice suggests that the class II molecule on the fibroblasts can present endogenously processed TSHR peptide to T cells. To identify TSHR epitopes presented by RT4.15HP-TSHR we tested spleen cells from hyperthyroid mice with overlapping peptides spanning the extracellular domain of the TSHR. TSHR 121– 140 peptide induced the highest proliferative response of spleen cells. Furthermore, TSHR 121–140 peptide-specific T cells proliferated well in response to RT4.15HP-TSHR but not to either RT4.15HP-pSG5 or DAP.3-TSHR, in the absence of the exogenous antigenic peptide. These data indicate that TSHR 121–140 peptide contains a naturally processed TSHR T cell epitope that is presented by class II moleculepositive fibroblasts. This peptide contains an I-Ak binding motif (Glutamic acid at anchor position 1 and Glycine at anchor position 9) [11,12], consistent with the conclusion that this is a T cell epitope in this mouse model of Graves' disease. Although, in general, the class II molecule is mainly involved in the presentation of exogenous antigens, it has been demonstrated that endogenous proteins can be presented to T cells by the class II molecule. This may especially be the case for transmembrane protein antigens since it has been reported that immunoglobulins [13,14], transferrin receptor, hepatitis B surface antigen [15], TPO, and TSHR [16] are bound to class II molecules and presented to T cells [17–19]. In this report, we describe an attempt to prevent experimental Graves' disease by pretreatment with a peptide that contains an immunodominant T cell epitope of the TSHR. It was reported in a collagen-induced arthritis model that low dose of intranasal administration of the immunodominant peptide of type II collagen might prime T cells, induce cytokines, and enhance the disease. Disease activity was correlated with the level of antigen-specific T cell activation [20]. In the present study the amount of peptide administered intranasally was 100 μg/dose, similar to that in reports showing suppression of the T cell response by intranasal administration of other peptides [5,21]. The finding that TSHR 121–140-specific T cell proliferation was reduced compared to control mice indicates that peptidespecific immune suppression was achieved in our experiments. This is also evidenced by the tendency toward suppression of the 3 cytokines measured. However, despite the down-regulation of the T cell response to this
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T. Arima et al.
Figure 5 Cytokine production of spleen cells stimulated with TSHR 121–140 peptide. IL-4 (A), IFN-gamma (B), and IL-10 (C) were assayed by ELISA in triplicate cell culture supernatants of spleen cells from three mice, each pretreated with TSHR 121–140 peptide or with PBS before intraperitoneal RT4.15HP-TSHR (TSHR+/class II+) fibroblast immunization. Mice were sacrificed 14 days after the last immunization, and spleen cells were challenged in vitro with TSHR 121–140 peptide. Data were presented as mean ± SEM. Intranasal pretreatment with TSHR 121–140 peptide caused a reduction of all the cytokines; the IFN-gamma reduction was statistically significant (P = 0.045).
immunodominant TSHR epitope, Graves' disease was distinctly enhanced both in frequency and severity by intranasal administration of the TSHR epitope peptide rather than prevented. Experimental autoimmune thyroiditis in murine models has been effectively suppressed by oral administration of either porcine [22] or human thyroglobulin [23]. In the former reports, type 2 CD8 T cells which produce IL-4 and TGF-beta mediated the suppression. In the latter experiments, both Th1 and Th2 responses were down-regulated. These data support the hypothesis that thyroglobulininduced thyroiditis is mediated by Th1 cells. In contrast, a Th2 response plays an important role in the development of hyperthyroidism in our mouse model of Graves' disease since it has been shown that alum and pertussis toxin enhanced disease expression, whereas complete Freund's adjuvant (CFA) delayed the disease [24]. Thus, the shift of Th1/2 balance to Th2 by intranasal administration of the TSHR peptide might be a cause of the increased frequency of disease expression and the enhancement of the serum T4 levels in TSHR 121–140 treated mice. However, in our experiments production of murine Th2 cytokines IL-4 and IL-10 by immune cells was not enhanced but rather suppressed by the intranasal administration of the TSHR peptide. Although suppression of IFN-gamma was statistically significant, the ratio of IL-4/ IFN-gamma or IL-10/IFN-gamma was not different between hyperthyroid and normothyroid mice (data not shown). Thus the mechanism of enhancement of hyperthyroidism by intranasal administration of an immunodominant epitope of TSHR in the present study remains to be determined. In conclusion, our present data suggest that mucosal administration of a peptide containing an immunodominant TSHR T cell epitope may aggravate or promote experimental Graves' disease rather than suppress its onset. Although the precise mechanism of enhancement of the disease remains to be determined, this should be taken into consideration when peptide-based immunotherapy for human Graves' disease is contemplated.
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