Reprints Available Directly from the Publisher Photocopying Permitted by License Only a Striational Muscle Antigen and Myasthenia Gravis- Associated Thymomas Share an Acetylcholine-receptor Epitope

The coincidence of autoantibodies against the acetylcholine receptor (AChR) and muscle striational antigens (SA) is a characteristic finding in thymoma-associated myasthenia gravis (MG), but their origins are still unresolved. Some common muscle antigens that were shown to be targets of anti-SA autoantibodies in thymoma-associated MG have also been detected in normal or neoplastic th)fmic epithelial cells, suggesting that the release of (eventually altered) antigens from the thymic tumors Could elicit SA autoimmunity. In contrast to this model, we report here that titin, which is a recently reported target of SA autoimmunity, is not expressed in thymomas. In addition, we show that skeletal muscle type-II fibers exhibit a striational immunoreactivity with monoclonal antibody mAb155, which was previously identified to label a very immunogenic cytoplasmic epitope of the AChR and neoplastic epithelial cells of MG-associated thymomas. We conclude from these findings that titin autoimmunity in thymoma-associated MG is either due to a molecular mimicry mechanism involving tumor antigens (other than titin) or is a secondary phenomenon following release of titin from muscle. Based on the common immunoreactivity of the AChR, a striational antigen and thymoma, we suggest as the pathogenetic mechanism of thymoma-associated MGa "circulus vitiosus" in which SA autoimmunity could help maintain the AChR autoimmunity that is primarily elicited by the thymomas.


INTRODUCTION
Myasthenia gravis (MG) is characterized by an abnormal fatiguability of muscle produced in most patients by autoantibodies against the nicotinic acetylcholine receptor (AChR). These autoantibodies are thought to impair neuromuscular transmission by reducing the number of muscle endplate AChR or blocking their function (Drachman, et al., 1980;Burges et al., 1990) In addition to anti-AChR autoimmunity, 70% of MG patients exhibit thymitis with or without *Corresponding author: Present address: Pathologisches Institut der Universit/it, Josef Schneider Str. 2, 8700 W(irzburg, Germany. lymphofollicular hyperplasia (M(iller-Hermelink et al., 1986;Kirchner et al., 1986) and 10% have a thymoma (Kirchner and MiJller-Hermelink, 1989). As an important diagnostic feature, only the latter group of MG patients almost invariably has high titers of heterogeneous autoantibodies against striational muscle antigens (SA) in addition to anti-AChR autoantibodies (Peers et al., 1977;Aarli et al., 1990;Connor et al., 1990;Ohta et al., 1990). In thymoma patients without MG anti-SA autoantibodies occur in only 24% (Williams and Lennon, 1987).
How SA autoimmunity is elicited in thymomas is not known. However, the finding that myosin and some less well-defined muscle proteins are anti-SA-autoantibody targets that also occur in 78 A. MARX et al. normal or neoplastic thymic epithelial cells (Gilhus et al., 1984;Williams and Lennon, 1987;Dardenne et al., 1987) has led to the hypothesis that thymomas might be the sites of SA autosensitization. Because an antigenic relationship between striational antigens and the AChR was not known, it is obscure whether autoimmune reactions against SA and AChR are interrelated.
Recently, titin was identified as a major target of anti-SA autoantibodies in MG (Aarli et al., 1990). In the present paper, we report that titin is not expressed in thymomas, suggesting that this kind of SA autoimmunity is either not due to intratumorous autosensitization or occurs by molecular mimicry mechanisms, that is, by titinrelated antigenic determinants in thymoma proteins that may cross-react with titin-"specific" T cells, which could induce an antititin autoantibody response when released to the periphery. In addition, an immunoreactivity is demonstrated that is shared by the AChR alpha subunit and a striational antigen different from titin. Because the respective epitopemcorresponding to AChR alpha371-378mis the only one so far detected in MG-associated thymomas, we suggest that AChR autoimmunity and part of SA autoimmunity in MG-associated thymomas could be interrelated.

Titin Is Expressed in Thymic Myoid Cells but Not in Thymomas
Frozen sections of two normal thymuses were probed with antititin mAb to three different titin epitopes. As shown in Fig. 1, immunoreactivity with all three mAbs was seen in a few round or spindle-shaped cells inside the medulla of normal thymuses close to Hassal's corpuscles. Both the localization and the morphology of these, cells are typical of thymic myoid cells (Kirchner et al., 1988). This was confirmed by double immunolabeling of thymic myoid cells by both antidesmin or anti-AChR mAb and by antititin mAbT12 (Figs. 2a and 2b). There was no expression of titin in keratin-positive nonneopalstic thymic epithelial cells (Fig. 2c).
Because of the high incidence of antititin autoantibodies typical of MG/thymoma patients (Aarli et al., 1990), we looked for an aberrant expression of titin in thymoma because the denovo expression of other proteins has previously been reported as a typical feature of neoplastic thymic epithelium Marx et al., 1990Marx et al., , 1991. However, with respect to the FIGURE 1. Immunoreactivity of a few round or spindle-shaped cells inside the human thymic medulla with antititin monoclonal antibodies T4 (a), T12 (b), and T32 (c). Morphology and localization are typical of thymic myoid cells (cf. Fig. 2). No immunoreactivity is encountered in epithelial cells. Immunoperoxidase technique on frozen sections (x160). STRIATIONAL AChR-EPITOPE IN MYASTHENIA 81 FIGURE 4. Immunoreactivity of a striational muscle antigen in about 50% of quadriceps muscle fibers with anti-AChR mAb155 directed against the AChR epitope alpha371-378 (a). No reactivity of muscle with anti-AChR mAb195 directed against the AChR "main immunogenic region" (Tzartos et al., 1983) (b). In contrast to the striational immunoreactivity of mAb155, the immunoreactivity with antititin mAb T12 is encountered in 100% of muscle fibers (c), demonstrating the nonidentity of titin and the AChR-epitope-bearing striational antigen in human quadriceps muscle (all sections #$361). Frozen sections, immunoperoxidase (x400). FIGURE 5. Immunoreactivity of anti-AChR mAb155 with a striational antigen is confined to type-II fibers in the quadriceps muscle. Serial cross sections labeled with mAb155 (a; immunoperoxidase) and reacted for ATPase activity at pH 9.4 (b). Immunoreactivity with mAb155 coincides with a high ATPase activity (dark color) typical of type-II fibers (Dubowitz, 1985) (x 400).
have to prove whether the striational antigen immunolabeled by mAb155 (Fig. 4a) is identical to the thymoma proteins bearing the AChR epitope detected by mAb 155   Quadriceps muscle +c aImmunoreactivity with anti-AChR mAb155 (Tzartos et al., 1986).
bImmunoreactivity of mAb155 with thymic myoid cells and medullary epithelial cells .
cStriational pattern in type-II fibers.
1990), and whether either molecule is in fact a target of autoimmune B or T cells in vivo. If the latter question could be positively answered, our finding suggests a pathogenetic model in which striational autoimmunity might help to maintain AChR autoimmunity. Such a model of a "circulus vitiosus" would explain the clinical experience that removal of the thymoma does not ameliorate the MG. and normal quadriceps muscle (obtained from autopsies within 6 hr after death) were investigated using cryostat sections from snap frozen tissue. Tumors were classified according to Kirchner and Mi.iller-Hermelink (1990). Clinical data of patients are summarized in Table 1. The diagnosis of generalized myasthenia gravis was based on clinical findings, including an abnormal muscle fatiguability, electrophysiological investigations and anti-AChR serum titers that were above I nmol/L.

Immunohistochemistry
The monoclonal antibodies (mAb) used in this study are described in Table 2. The immunohistochemical three-stage immunoperoxidase technique and the double-labeling procedure combining the immunoperoxidase with an alkaline phosphatase technique (as the first and second step, respectively) were as described previously, including the same control experiments (Kirchner et al., 1988).

Patients and Tissues
Six thymomas from MG patients, two normal thymuses (obtained during thoracic surgery), To investigate whether skeletal muscle immunoreactivity with anti-AChR mAb155 was confined to either type-I or -II muscle fibers serial frozen sections were processed for routine enzyme histochemical determination of ATPase activity at both pH 4.6 and pH 9.4 (Dubowitz, 1985).