Thymic myoid cells as a myasthenogenic antigen and antigen-presenting cells

Journal of Neuroimmunology 150 (2004) 80 – 87 www.elsevier.com/locate/jneuroim

Thymic myoid cells as a myasthenogenic antigen and antigen-presenting cells Megumi Y. Matsumoto a, Hidenori Matsuo b,c,*, Takashi Oka d, Takayasu Fukudome b,c, Kazuhiro Hayashi d, Hirokazu Shiraishi e, Masakatsu Motomura e, Noritoshi Shibuya c, Hiroyoshi Ayabe a a

Division of Surgical Oncology, Department of Translational Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan b Division of Clinical Research, Kawatana National Hospital, Nagasaki, Japan c Department of Neurology, Kawatana National Hospital, Nagasaki, Japan d Department of Pathology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan e First Department of Internal Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan Received 27 August 2003; received in revised form 12 December 2003; accepted 23 January 2004

Abstract We investigated immune property of a myoid cell line, established from Fisher rat thymus. Immunization of syngeneic rats with the myoid cells induced anti-rat acetylcholine receptor (AChR). Implantation of them into the thymus failed to induce typical thymic pathology of human myasthenia gravis (MG) or anti-AChR responses. We also demonstrated that the myoid cells were able to present exogenous antigens to T cells and induce antigen-specific T cell proliferation. These results suggest that myoid cells have the potential antigenicity to induce antiAChR and the functions of antigen-presenting cells, but their expansion in the thymus may not directly cause MG. D 2004 Elsevier B.V. All rights reserved. Keywords: Myasthenia gravis; Myoid cells; Thymus; Germinal center; Autoantibody

1. Introduction Myasthenia gravis (MG) is an autoimmune disease in which autoantibodies directed to the nicotinic acetylcholine receptor (AChR) impair neuromuscular transmission. These antibodies cause loss of AChRs at the motor end-plates and subsequent muscle weakness (Drachman, 1994). The production of these IgG antibodies is T cell-dependent in experimental animals and probably in human MG too, and the initial sensitization of the specific helper T cells is widely believed to be a crucial step in the induction of autoimmunity. Despite suggestions that viral or bacterial infections may trigger human MG (Aoki et al., 1985; Schwimmbeck et al., 1989; Stefansson et al., 1985), the * Corresponding author. Division of Clinical Research and Department of Neurology, Kawatana National Hospital, Shimogumi-gou 2005-1, Kawatana, Higashisonogi-gun, Nagasaki 859-3615, Japan. Tel.: +81-956823121x2003; fax: +81-956-833710. E-mail address: [email protected] (H. Matsuo). 0165-5728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.01.022

molecular or cellular events leading to antibody production against neuromuscular junction components remains uncertain(Drachman, 1994). The high affinity and specificity of the MG anti-AChR response measured in vitro, and its similarity to that occurring in animals immunized against purified AChR, have supported the idea that MG is induced by self-AChR rather than by a cross-reaction (Vincent et al., 1998). If so, the immune cells which lead to production of the autoantibody must encounter (and be primed with) native AChR in vivo. The thymus is another focus in MG, where scarce AChR is expressed sparsely. MG is frequently associated with thymic abnormalities: thymic hyperplasia and thymoma (Castleman and Norris, 1949; Drachman, 1994). Therefore, the thymus has been implicated as a possible site of origin that triggers autoimmunity against AChR (Drachman, 1994; Vincent et al., 1998). Indeed, previous studies have demonstrated production of antibodies against AChR by thymic lymphocytes in vitro (Newsom-Davis et al., 1981) and apparent clinical benefit of thymectomy

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(Simpson, 1958; Rowland, 1987). In the thymus, there are AChRs expressed on myoid cells (Kao and Drachman, 1977; Schluep et al., 1987) and thymic epithelial cells (Wakkach et al., 1996). There are also many mature T and B lymphocytes and professional antigen-presenting cells (APCs), such as dendritic cells (Newsom-Davis et al., 1981; Bofill et al., 1985; Kirchner et al., 1988). Patients with MG have AChR-specific T lymphocytes in the thymus (Melms et al., 1988). Since investigators found thymic muscle cells bear AChRs (Kao and Drachman, 1977), myoid cells have been thought to trigger the autoimmune response in MG(Wekerle and Ketelsen, 1977; Kirchner et al., 1988). The thymic medulla of most patients with early-onset MG shows lymph node-like T cell infiltration and germinal center (GC) formation (Castleman and Norris, 1949; Bofill et al., 1985; Kirchner et al., 1988), and thymic myoid cells do exist there (Bofill et al., 1985; Schluep et al., 1987; Kirchner et al., 1988). While AChR on myoid cells would therefore be involved in the pathogenesis of MG, the supporting evidence is indirect (Sommer et al., 1990; Roxanis et al., 2001, 2002), and there is no clear evidence for an initiating role or an ability to prime T cells. To study the antigenicity of myoid cells, we implanted a thymic myoid cell line into syngeneic rat thymus, or immunized the animals with myoid cells, and assessed whether antibodies to AChR, which play a major role in the development of clinical symptoms of experimental autoimmune myasthenia gravis (EAMG), are induced or not. Furthermore, because it is not known whether myoid cells function as an APC, as do so myoblasts (Goebels et al., 1992; Curnow et al., 2001), we investigated their functional capacity to present antigens (Ags) to T cells using syngeneic T cell lines.

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acetylcholine receptor on the 14th day. Finally, these cells started to contract spontaneously. In several experiments, ST1B cells were cultured in the presence of 500U/ml recombinant rat interferon-g (IFN-g ; Genzyme/Techne, USA) for 72 h to induce class II antigens (Hohlfeld and Engel, 1991; Goebels et al., 1992; Baggi et al., 1993; Curnow et al., 2001). 2.2. Injection of myoid cells We immunized 6-week-old female Fisher rats (F344/ DuCrj; Charles River Japan, Yokohama, Japan) with 5  106 or 1  107 ST1B in Freund’s complete adjuvant (CFA) in the hind footpads once or three times in 2 – 3 week intervals. In another series, we implanted 1  106 or 5  106 ST1B cells, treated with or without IFN-g for 72 h, into the thymus of 6-week-old female Fisher rats. Intrathymic injection was performed by exposing the thymus gland through a small incision above the sternum. In some experiments, ST1B cells were cultured with pooled anti-AChR rat serum (a final concentration 10%) taken from chronic EAMG rats for 6 h at 37 jC, or with mitomycin C (MMC, 40 Ag/ml) for 4h before the thymic injection. Every week after immunization, rats were weighed and observed for clinical signs of EAMG according to the scale reported (Lennon et al., 1975). We took blood samples at several points, then removed the thymuses for histological examination. Animal experiments were conducted under the guidelines of the Animal Care and Use Committee of Nagasaki University. During the observation period, rats were housed under standard approved conditions. 2.3. Anti-AChR antibody assays

2. Materials and methods 2.1. Thymic myoid cells A thymic myoid cell line was isolated as described elsewhere (Oka et al., 2000). Briefly, it was established by the cocultivation of Fisher rat thymic cells with Human Tlymphotropic virus type-I (HTLV-I)-producing human lymphoid cells. Cloned myoid cell precursor cells (ST1B) were grown in Dulbecco’s modified Eagle’s medium (DMEM) and 5% fetal calf serum (FCS), and passaged with 1% trypsin/EDTA every 3 days. These cells differentiate to myoid cells both in vitro and in vivo and also retain some physiologically normal phenotypes. No integration of HTLV-I was detected in them by Southern blot hybridization. Multinucleated giant cells appeared on the 4th day and formed myotube-like structures with Z-band-like composition, sarcomeric structures and tubular honeycomb arrays accompanied by induced expression of MyoD1 and the various muscle specific antigens including a-sarcomeric actin, skeletal muscle myosin, myoglobin, desmin and

The blood sample was allowed to clot, spun for 10 min and the resulting serum was stored at 70 jC until use. Measurement of antibody to rat AChR was performed by a double-antibody radioimmunoassay, using 125I-a-bungarotoxin(Lindstrom et al., 1976). A crude extract of rat muscle AChR prepared from denervated muscles of normal rats was used as antigen to detect the autoantibody. All assays were performed in triplicate. 2.4. Antigens AChR protein was extracted from the electric organs of Narke Japonica using Triton X-100 and purified by affinity chromatography on a-cobratoxin-agarose resin (Eldefrawi and Eldefrawi, 1973). A synthetic peptide corresponding to the rat-AChR a128 – 142 was used as a stimulating Ag for AChR-specific T cells. Myelin basic protein (MBP) and ovalbumin (OVA) were purchased from Sigma Chemical (USA). Purified protein derivative of tuberculin (PPD) was obtained from Japan B.C.G. (Tokyo).

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2.5. Production of Ag-specific T cell lines Ag-specific T cell lines were isolated from the draining regional lymph nodes of Fisher rats immunized in the footpads (0.2 ml total) bilaterally with MBP, OVA, PPD or Narke-AChR, respectively, in CFA. Lymph node cells were cultured with relevant Ag in 200 Al of RPMI 1640 (GIBCO BRL), supplemented with 100 U/ml penicillin, 100 Ag/ml streptomycin, 1 mM L-glutamine, 5  10 5 M 2-mercaptoethanol (ME), and 1% fresh syngeneic serum for 3 days. Responding lymphoblasts were isolated on a Ficoll density gradient and expanded further in a growth medium containing 10% FCS, antibiotics, L-glutamine, 2-ME as above, and 5% supernatant of 48 h rat splenocyte cultures with Concanavalin A (Con A, from Sigma Chemical USA) (rat growth factor). The line cells were propagated with syngeneic MMCtreated thymocytes, relevant Ag, and subsequent rat growth factor. 2.6. Proliferation assays In order to measure Ag-specific T cell proliferation, ST1B cells treated with or without INF-g were cultured with MMC (40 Ag/ml) for 2 h at 37 jC, washed extensively and plated into flat-bottomed microtiter wells at 1  104 cell/ well. 5  104 Ag-specific T cells were added and then incubated with or without the relevant Ags for the proliferation assay. Quadruplicate cultures with 200 Al stimulation medium were set up with or without Ag (10 Ag/ml). Con A was used as a positive control at a concentration of 2.0 Ag/ml. After 72 h of culture at 37 jC in 5% CO2, cultures were pulsed with the thymidine analogue, 5-bromo2V-deoxyuridine (BrdU) for 2 h, and proliferative responses were quantified with the Cell Proliferation ELISA kit (Boehringer Mannheim, Germany). The resultant color development is proportional to the concentration BrdU in the DNA synthesizing cells in each microwell. Absorbances were measured with an optical densitometer. 2  106/well syngeneic MMC-treated thymocytes were used as APC for control.

controls immunized with CFA alone were killed and a portion of the diaphragm with its motor nerve was removed and mounted in a chamber continuously perfused with oxygenated (95% O2, 5% CO2) Tyrode solution. A Dtubocurarine chloride was used at a concentration sufficient to inhibit muscle contraction. Miniature end-plate potentials (MEPPs), end-plate potentials (EPPs) and resting membrane potentials (RMPs) were recorded at 29.5 F 0.5 jC with an MEZ-8201amplifier (Nihon Kohden, Japan). Potentials were corrected to a standard resting membrane potential of 80 mV assuming an equilibrium potential of 0 mV. 2.9. Histopathological studies Thymuses taken at each point were fixed with 4% paraformaldehyde. Paraffin sections of the thymus were stained with hematoxylin and eosin (H and E). Some specimens were stained with anti-desmin (BioScience Products, Emmenbrucke, Switzerland) or anti-cytokeratin (CAM5.2; Becton Dickinson, San Jose,CA) and the secondary antibodies.

3. Results 3.1. Immunization with myoid cells A single immunization with myoid cells (1  107) in CFA in foot pads of Fisher rats induced relatively low titers

2.7. IFN-g assays 100 Al of supernatant was removed from each well for cytokine ELISA after 24 h of culture. Production of IFN-g was evaluated with an ELISA kit (TFB, Tokyo, Japan), following the manufacturer’s protocol. Cytokine production was calculated from a standard curve of the corresponding concentration of recombinant rat IFN-g. 2.8. Electrophysiological studies For conventional intracellular microelectrode study, 4 rats immunized 3 times with ST1B 5  106/CFA and 4

Fig. 1. Production of anti-AChR antibodies after immunization with myoid cells in Freund’s complete adjuvant (CFA). Mean titers of eight rats per group are plotted. 5 M, 10 M: rats immunized 5  106 or 10  106 ST1B cells in CFA. Bars are S.D. of each group. The cutoff value for positivity is 30 pmol/l.

M.Y. Matsumoto et al. / Journal of Neuroimmunology 150 (2004) 80–87 Table 1 The production of anti-acetylcholine receptor (AChR) antibodies in serum after immuniztion with the myoid cell line Anti-AChR titer (pmol/l) Rats immunized with myoid cells 3 weeks after the third immunization (n = 9) Chronic EAMG pooled seruma Negative control

127.1 F 67.1 (from 31.4 to 205.0) 5780.7 < 30

a

Pooled serum from experimental myasthenia gravis rats in chronic phase: Lewis rats were immunized with Narke-acetylcholine receptor/ Freund’s complete adjuvant.

of anti-AChR antibodies 4 weeks post-immunization (Fig. 1). Repeated immunization with myoid cells also elicited anti-AChR in all rats immunized (Table 1). The anti-AChR titers remained much lower than those of chronic EAMG induced by a single immunization of Narke-AChR in Lewis rats. No rat showed apparent weakness, weight loss, or other clinical signs indicative of EAMG. Electrophysiological studies demonstrated that the amplitudes of the MEPPs were reduced (0.66 F 0.02 mV, n = 4) and the quantum contents of EPPs (41.7 F 2.6, n = 4) were increased in rats immunized with ST1B compared with those of the controls (MEPPs: 0.69 F 0.02 mV; n = 4, EPPs: 39.4 F 1.9, n = 4) although neither reached statistical significance. Histological examinations 4 weeks after the 3rd immunization revealed mild atrophy and swelling of

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perithymic LNs, but there was no thymic hyperplasia or GC formation. 3.2. Effects of intrathymic injection of myoid cells Next we investigated whether intrathymic injection of myoid cells lead to development of clinical EAMG. In these experiments, 1  106 or 5  106 myoid cells (ST1B) were injected intrathymically. No rat exhibited typical clinical signs of EAMG up to 6 weeks after the injection, but a few rats injected with 5  106 ST1B showed weight loss, probably due to thymic enlargement. Injection of ST1B with pretreatment with interferon-g, MMC, or chronic EAMG rat serum also failed to induce clinical EAMG. Moreover, anti-rat AChR antibodies did not rise ( < 30 pmol/L) in the sera from any rats implanted intrathymically with ST1B up to 6 weeks after the injection. In histologic investigation of the thymus 8 weeks after implantation, we found that the implanted cells highly differentiated into the striated muscles in the thymus, as confirmed by staining with anti-desmin (Fig. 2) and smooth-muscle actin. In rats injected 5  106 ST1B, we found that the myoepithelial cells formed tumor-like mass in the thymus. There were no other changes in the surrounding tissue including GC formation. Interestingly, we observed that Hassall’s corpuscles increased in the vicinity of the implanted lesions in thymus from 1 of 6

Fig. 2. Implantation of myoid (ST1B) cells into adult Fisher rat thymus. ST1B cells differentiated to skeletal muscle cells (arrow heads) and cytokeratinpositive epithelial cells (arrows) forming characteristic Hassall’s corpuscles at the injected site. A, C: hematoxylin – eosin staining; B: immuno-staining with anti-cytokeratin; D: immuno-staining with anti-desmin. Bars: 100 Am (A, B); 10 Am (C, D).

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rats injected with 1  106 ST1B after IFNg treatment, suggesting some of the myoid cells differentiated into epithelial cells in the thymus. Indeed, ST1B cells differentiate to cytokeratin-positive epithelial cells or Hassall’s corpuscles close to the muscle cells differentiated from ST1B (Fig. 2A,B). To test the possibility that phagocytosis of material from degenerating myoid cells by thymic elements may play a role in sensitization, we injected 5  106 ST1B cells treated with MMC (40 Ag/ml) for 4 h into the thymus of four rats. In this group, no rat developed clinical EAMG and no antiAChR was detected until 6 weeks after implantation. Next, we intrathymically injected 1  106 ST1B cells cultured with anti-AChR serum (10%) after IFN-g treatment. None of four rats in this group developed EAMG and no antiAChR was detected until 6 weeks. Histologic examination of the thymuses in these groups did not reveal any GC formation.

3.3. Ag-presenting capacity of the thymic myoid cells We confirmed, using several Ag-specific T cell lines, that the MMC-treated ST1B were able to present the exogenous Ags (peptide a128 – 142, MBP, OVA and PPD) to the relevant T cells and induced Ag-specific T cell proliferation as measured by the incorporation of BrdU (Fig. 3). The Ag presentation usually needed induction of class II expression with IFN-g treatment, but one line occasionally without IFN-g treatment (Fig. 3a). Next we tested whether the myoid cells were able to present the epitope from their endogenous AChR to the AChR peptide-specific T cell line (SH). We first confirmed that the line SH responded to rat AChR, recombinant human AChR a-subunit (kind gift from Prof. N. Willcox) and Narke-AChR as well as peptide a128 – 142 (data not shown). While MMC-treated ST1B cells did not directly induce detectable Ag-specific proliferation of SH, there was

Fig. 3. Antigen (Ag)-induced proliferation of syngeneic Ag-specific T cell lines in the presence of myoid cells (ST1B, mitomycin C-treated) or thymocytes. (a) AChR peptide-specific T cell line; (b) myelin basic protein (MBP)-specific T cell line, (c) ovalbumin (OVA)-specific T cell line; (d) purified protein derivative of tuberculin (PPD)-specific T cell line. The myoid cells were cultured with (+) or without ( ) interferon-g (IFNg), then treated with mitomycin C for 2 h at 37 jC, washed extensively and plated at 1  104 cell/well. APC: antigen-presenting cells; Thx: MMC-treated thymocytes; pep: AChR peptide (a128 – 142, 10 Ag/ ml). OVA, MBP and PPD were used at 10 Ag/ml. Concanavalin A (Con A) was used as a positive control at 2.0 Ag/ml. All results were in quadruplicate with S.D. of < 10%. Data are representative of at least two experiments.

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Fig. 4. The proliferative responses (a) and interferon-g secretion (b) of AChR peptide-specific T cells to the myoid cells. (a) The myoid cells (ST1B) were cultured with interferon-g, then treated with mitomycin C, washed extensively and plated at 1  104 cell/well (ST1B) or 2  104 cell/well (2 ST1B). MMCtreated thymocytes (Thx) and AChR peptide (Ag: a128 – 142, 10 Ag/ml) were added in the positive control. The results of proliferation assay were in quadruplicate with S.D. of < 10%. Data are representative of three experiments. (b) Interferon-g secretion by acetylcholine receptor-peptide (SH) and ovalbumin (OVA)-specific T cells were measured following presentation of antigen. LNx: MMC-treated syngeneic lymph node cells; Ag: peptide a128 – 142 or OVA at 10 Ag/ml. The results were in triplicate with S.D. of < 10%. Data are representative of two experiments.

a weak but significant IFN-g response in the absence of added AChR (Fig. 4).

4. Discussion We here demonstrate that (1) antibodies to self-AChR can be induced by immunization with thymic myoid cells as Ag, (2) the expansion of myoid cells in the thymus does not elicit detectable anti-AChR response, and (3) myoid cells can present exogenous Ags to the Ag-specific T cells, and induce T cell proliferation. These findings may help to elucidate the autoimmune processes occurring in the thymus of patients with MG. It has been suggested that myoid cells in the thymus might be involved in autosensitization (Van de Velde and Friedman, 1966; Kao and Drachman, 1977; Wekerle and Ketelsen, 1977; Kirchner et al., 1988; Roxanis et al., 2002). AChR has a striking immunogenicity: previous studies (Scadding et al., 1986; Jermy et al., 1989, 1993) have proved that minute amounts of AChR are able to induce anti-AChR response in syngeneic animals without adjuvant. In the present study, we found that immunization with myoid cells with CFA can induce anti-AChR antibody production, probably in dose-dependent manner. A single immunization with 5  106 myoid cells failed to induce antiAChR. This may be because the amounts of AChR on the myoid cells were too small to elicit antibody production by a single immunization. The pathogenicity of these antibodies is not clear because they induced very few clinical symptoms of EAMG with equivocal electrophysiological neuromuscular impairment. Major reason is that the titers of anti-

AChR were relatively low compared to those in Lewis rats immunized with AChR from electric organs of eels or rays. Previous studies in which rodents immunized with small amount of mammalian AChR or AChR peptide also produced relatively minor immune responses and less prominent myasthenic symptoms (Lennon et al., 1985, 1991; Jermy et al., 1993). In the present study, we show, for the first time, that thymic myoid cells are able to function as APC. They are fully able to present various exogenous Ags, including protein Ags, and induced Ag-specific T cell proliferation. These findings imply that they have ability of Ag processing and adequate co-stimulatory capacities to induce T cell proliferation. The Ag presentation by the myoid cells often needed class II induction by IFN-g treatment. We confirmed that IFN-g induced class II expression in myoid cells (data not shown), as in myoblasts (Hohlfeld and Engel, 1991). In some experiments, the myoid cells presented Ag to the relevant T cells without IFN-g treatment (Fig. 3a). This may be consistent with the previous observation that some ST1B cells weakly expressed class II in culture (Oka et al., 2000). Low levels of class II have been detected on about 15% of myoid cells in the normal and MG thymus (Roxanis et al., 2000). Perhaps also, IFN-g secreted by T cells responding early to peptide presented by class II positive myoid or T cells induce or up-regulate class II expression on adjacent myoid cells. Therefore, myoid cells can be a target of autoimmune attack and involved in the pathogenesis of human MG. On the other hand, we failed to demonstrate that the thymic myoid cells were able to present endogenous Ag and induce T cell proliferation. This may also be due to small

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amount of endogenous AChR on the myoid cells, and/or a low turnover. The AChR-specific T cell line we used was established by using rather high concentration of peptide as a stimulating Ag after immunizing with Narke-AChR. There is a single substitution 139E X Q in the sequence of AChR a128– 142 region between rat and electric ray (Noda et al., 1982; Witzemann et al., 1990). So, the T cells may not be sensitive enough to recognize such epitope from a minute amount of AChR on myoid cells. Myoblasts were able to present an endogenous epitope to a CD4+AChR-specific T cell clone (Curnow et al., 2001), which was originally isolated from the hyperplastic thymus of a MG patient and responded to exogenous Ag at a very low concentration(Ong et al., 1991). They (Curnow et al., 2001) observed T cell activation, resulting in secretion of IFN-g, but no proliferation, and speculated that this phenomenon may attribute to lack of conventional co-stimulatory molecules. As mentioned above, the myoid cells we used have enough co-stimulatory molecules to proliferate T cells. Therefore, we speculate that the major reason for which the AChRspecific T cell failed to proliferate may be the small quantity of AChR on the myoid cells. Myoid cells reportedly label for cytokeratins (Schluep et al., 1987). Strikingly, some of the implanted myoid cells differentiate into epithelial cells in the thymus. A previous study (Zoltowska et al., 1998) also reported that myoid cells existed in the vicinity of Hassall ’s corpuscles and within them in humans, suggesting that myoid cells are source of some thymic epithelial cells. Furthermore, another investigator (Itoh, 1983) predicted that the origin of myoid cells might be pluripotent stem cells, when he cloned them from the rat thymus. These observations may shed new light on the development and differentiation of cellular components of the thymus. The exact mechanisms leading to their differentiation, including roles of cytokines, remain to be investigated. The role of thymic myoid cells in the pathogenesis of MG remains unclear. Certainly myoid cells can induce anti-AChR responses, as shown here, and some of them expressed MHC-class II and could be involved in the autoimmune process. However, their expansion in the thymus may not cause MG as demonstrated in the present study, which is accordance with the previous report that a patient with rare myoid thymoma did not have MG symptoms (Henry, 1972). Injection of MHC-class II-induced myoid cells by IFN-g also failed to trigger autoimmune process in the thymus. There have been two major hypotheses on the role of thymic abnormality in MG. Wekerle and Ketelsen (1977), Kirchner et al. (1988) and Marx et al. (1997) proposed that the AChR expressed on thymic myoid cells is the original autosensitizing Ag and the thymic changes in MG are primary events in the autoimmune pathogenesis of the disease. Others (Sommer et al., 1990; Utsugisawa et al., 2000; Roxanis et al., 2001, 2002) suggest that the hyperplastic features in MG thymus are secondary phenomena; the AChR-specific T cells are

selectively trapped and restimulated in the thymus after prior sensitization elsewhere and thymic myoid cells can be targeted by these T cells or the antibodies that they evoke. Meinl et al. (1991) tried to address this question by studying the thymuses of Lewis rats with EAMG. Major limitation in their approach is that T cells primed with Torpedo-AChR are rarely reactive to rat-AChR (Hohlfeld et al., 1981). Our finding that repeated immunization with myoid cells induced anti-AChR antibody implies that there must be rat-AChR-specific or myoid cell-reactive helper T cells and B cells in vivo. Nevertheless, these rats showed none of the typical MG thymic abnormalities. These results may support the previous observation by Meinl et al. Roxanis et al. (2002) proposed a hypothesis that an early autoantibody attack on myoid cells provokes local GC formation. Accordingly, we treated the myoid cells with anti-AChR serum before injection into the thymus but failed to induce GC formation in the thymus. These results suggest that additional mechanisms, such as particular inflammatory cytokines, hyperactive dendritic cells or breakdown of immnoregulation in the thymus, would be necessary to induce GC formation. If myoid cells are involved in initiating autoimmune responses in MG, that might require some species’ difference or danger signal (e.g. inflammation or complement activation). However, they could be involved subsequently because of their expression of whole AChR and MHC-class II (in some cells) and their Ag-presenting capacity. Acknowledgements We would like to thank Professor N Willcox (University of Oxford) for providing recombinant AChR and his helpful advice on the manuscript. This work was supported by a grant from the Neuroimmunological Disorders Research Committee of the Ministry of Health, Labor and Welfare of Japan. We wish to dedicate this publication to Professor Hiroyoshi Ayabe, who died suddenly after completion of this study. References Aoki, T., Drachman, D.B., Asher, D.M., Gibbs, C.J., Bahmanyar, S., Wolinskiy, J.S., 1985. Attempts to implicate viruses in myasthenia gravis. Neurology 35, 185 – 192. Baggi, F., Nicolle, M., Vincent, A., Matsuo, H., Willcox, N., NewsomDavis, J., 1993. Presentation of endogenous acetylcholine receptor epitope by an MHC class II-transfected human muscle cell line to a specific CD4+ T cell clone from a myasthenia gravis patient. J. Neuroimmunol. 46, 57 – 65. Bofill, M., Janossy, G., Willcox, N., Chilosi, M., Trejdosiewicz, L.K., Newsom-Davis, J., 1985. Microenvironments in the normal thymus and the thymus in myasthenia gravis. Am. J. Pathol. 119, 462 – 473. Castleman, B., Norris, E.H., 1949. The pathology of the thymus gland in myasthenia gravis: a study of 35 cases. Medicine 28, 27 – 58. Curnow, J., Corlett, L., Willcox, N., Vincent, A., 2001. Presentation by myoblasts of an epitope from endogenous acetylcholine receptor indi-

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