Myasthenia gravis 9 1988 S. Karger AG, Basel 0257-277X/88f0073-018952.75/0

Immunol Res 1988;7:t 89-199

Cellular Aspects of Myasthenia gravis I S. Berrih-Aknin a, S. Cohen-Kaminsky a, D. Neumann b, D. Safar a, B. Eymard a, C. Gaud a, P. Levasseur a, S. Fuchs b, J.-F. Bach c aCentre Chirurgical Marie-Lannelongue,CNRS UA 1159, ke Plessis-Robinson, France; bDepartment of Chemical Immunology,The Weizmann Institute of Science, Rehovot, Israel; cINSERM U25, H6pital Necker, Paris, France

The thymus and its cellular products, the T cells, are at play in many aspects of autoimmune diseases for which alterations in the function and number of immunoregulatory T cells are commonly found. Evidence that the production ofautoantibodies results from disturbance of regulatory T cells has been largely reviewed [1]. However, some diseases are associated with depressed suppressor T cell function while others are characterized by an increased helper T cell function [reviewed in 2]. These contrasting alterations may explain why thymectomy shows diverse effects on the course of experimental autoimmune diseases. Indeed, most experimentally induced autoimmune diseases, as well as lupus in MRL/1 mice and diabetes in NOD mice, are prevented by neonatal thymectomy; conversely, thymus ablation aggravates the lupus of NZB, (NZB • NZW)F1 and B • SB mice and the thyroiditis of the obese strain chickens [3, 4]. Myasthenia gravis (MG) is an autoimmune disease associated with autoantibodies directed against acetylcholine receptors This work was supported by grants from AFM, CNRS and CNAMTS-INSERM.

(AChR). A close relationship between disease pathogenesis and thymus function has been suggested by several lines of evidence. Morphological abnormalities of the thymus gland (hyperplasia or thymoma) are frequently found [5, 6], thymectomy has been reported to improve disease symptoms [7, 8], thymocytes from myasthenics produce spontaneously anti-AChR antibodies [9, I0] and their epithelial cells synthesize increased amounts of thymic hormones [11], one of which, thymopoietin, has been reported to depress neuromuscular conduction [12], to bind to AChR with a high affinity [13, 14] and to enhance the desensitization of the AChR under certain conditions [15]. Lastly, the presence of an AChR-like antigen in extracts of myasthenic thymi has been demonstrated both by its function (a-bungarotoxin binding) and its antigenicity (cross-immunoreactivity with muscle AChR) [16, 17]. Several mechanisms of action of autoantibodies are known to be involved: blockade of acetylcholine-binding to AChR, complement-mediated destruction of the junctional folds and antigenic modulation [18, 19]. Conversely, early events leading to autoantibody production remain enigmatic. The in-

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v o l v e m e n t o f c e l l u l a r m e c h a n i s m s has b e e n s u g g e s t e d by d a t a d e m o n s t r a t i n g m a n y T cell a b n o r m a l i t i e s [ r e v i e w e d in 20], b u t no c l e a r a s s o c i a t i o n with c l i n i c a l features h a s b e e n d e s c r i b e d . T h e d e v e l o p m e n t o f new t o o l s for s t u d y i n g cell a c t i v a t i o n led us to i n v e s t i g a t e antigen-specific and antigen nonspecific T cell r e s p o n s e s in M G . Three experimental approaches have been developed: immunohistological study o f t h e t h y m u s g l a n d in M G patients; e v a l u a tion of the activation status of thymic and p e r i p h e r a l b l o o d l y m p h o c y t e s (PBL) by testing t h e i r a b i l i t y to r e s p o n d to r e c o m b i n a n t IL2 (r-IL2) w i t h o u t p r e v i o u s s t i m u l a t i o n ; a n a l y s i s o f P B L p r o l i f e r a t i v e r e s p o n s e s to s y n t h e t i c p e p t i d e s c o r r e s p o n d i n g to s e l e c t e d d o m a i n s o f the A C h R a - s u b u n i t , in o r d e r to define some epitopes of the AChR involved in M G a u t o i m m u n e response.

Patients and Methods Patients MG thymocytes were mechanically extracted from thymic tissue obtained from patients thymectomized at Marie-Lannelongue Hospital (Le PlessisRobinson, France). Normal human thymic fragments used as controls were obtained from subjects undergoing surgery for cardiac lesions. Blood lymphocytes from myasthenic patients were collected at Marie-Lannelongue Hospital and isolated using Ficoll-Hypaque density gradient. Controls were volunteers blood donors. Disease severity was graded according to Osserman's classification (I: ocular symptoms; IIA: generalized form without bulbar symptoms; IIB: generalized form with bulbar symptoms). Immunofluorescence Studies on Frozen Sections Thymic fragments were frozen in liquid nitrogen just after surgery. Several antibodies were used: goat antihuman lgM antiserum coupled to FITC was pro-

vided by Dako; mouse monoclonal antifibronectin antibody (lmmunotech) revealed by SAM/Ig/TRITC (Cappel) was used in double labeling experiments. Labeling with rabbit antikeratin antibody (Dako) was revealed by SAR/Ig/FITC (Cappel). Unfixed frozen sections (2 lain thick) were processed as described below. For single labeling experiments, sections were incubated with the first layerspecific antibody for 15 rain and washed twice in PBS for 5 rain. If necessary, they were then subjected to the appropriate fluorescent conjugate for 15rain, washed again in PBS and mounted in glycerol/PBS. When double-labeling experiments were performed, the specimens were sequentially incubated with the first specific antibody and its proper fluorescent conjugate, followed by the second specific antibody and its corresponding conjugate. Evaluation of Responsiveness to r-IL2 Freshly isolated lymphocytes were cultured at concentrations of 5 X 10 6 cells/ml for thymocytes or 1 • 106 cells/ml for PBL in fiat-bottomed microtest plates in RPMI-1640 medium supplemented with 2 % AB human serum, r-IL2 (produced after genetic recombination in Escherichia coil; Boehringer) was added at serial doses from 3 to 50 U/ml. Cultures were set up in quadruplicate and maintained for 2-12 days. Proliferation was then measured by 3H-thymidine (CEA, France) incorporation (1 ~tCi/well). To test the specificity of the response, a monoclonal antiIL2 receptor blocking antibody (IOTI4a, Immunotech) was added to the cultures at serial doses from 0.1 to 100 riM. Proliferation Assay to Synthetic Peptides Synthesis of peptides corresponding to residues 169-181, 185-196, 330-340, 351-368 and 394--409 of torpedo or human AChR ct-subunit is described elsewhere [21-23]. Freshly isolated lymphocytes from 34 patients and 17 controls were cultured at concentrations of 1 X 106 eells/ml, in fiat-bottomed microtest plates in RPMI-1640 medium supplemented with 2 % AB human serum and various AChR peptides. In preliminary experiments serial doses of peptides from 0.1 to 10 lag/ml were tested. Optimal concentration was determirted as 5 lag/ml and used in all experiments. Cultures were set up in triplicate and maintained for 3-8 days. The maximal response was observed in 6-day cultures. Proliferation was then measured by a 20-

Cellular Aspects of Myasthenia gravis

191

Fig. 1. Low magnification of normal (a) and MG hyperplasia (h) thymus frozen sections stained with antikeratin antibody revealed by GAR/Ig/FITC. • 117. Note the clear corticomedullary differentiation in the normal thymus and the large keratin negative areas in hyperplasia.

hour 3H-thymidine (CEA) incorporation test (1 ~Ci/well). The results are expressed as proliferation index defined as follows: index = cpm with peptide/cpm without peptide. Responses higher than mean value + 2.6 SD of the 17 controls for a peptide were considered as positive.

StatisticalAnalysis Nonparametric Mann-Whitney test was used for small groups of patients (n < 30) and Student's t test was applied for larger groups (n > 30).

Results and Discussion

Immunohistological Studies of Thymic Hyperplasia T h y m i c hyperplasia is commonly defined in M G by the presence of germinal centers not necessarily associated with true global hypertrophy o f the gland. Such germinal centers may also be found in the normal thymus [24], but in smaller number. They are also found in several other autoimmune diseases [25, 26]. It has been reported [27] that the presence of many germinal centers is associated with a favorable clinical course, but opposite findings have also been published

[28]. In a recent study, we showed that thymic hyperplasia is generally observed in young patients with high anti-AChR Ab trier [29]. We investigated the thymic lymphoid and nonlymphoid compartments by immunofluorescence using several monoclonal and polyclonal antibodies in single and double labeling techniques on serial sections. In the normal thymus, the corticomedullary differentiation was clearly observed, while in M G thymi the epithelial network was highly compressed and dense clusters of keratin-positive cells including small Hassall's corpuscles were consistently seen (fig. 1). A major and unusual connective structure was identified, appearing as a framework of fibers often emerging from the inside of the thymic septae and entering into the interior o f the lobules. At the emergence sites, the basement membrane demonstrated discontinuities which were in contrast to the continuous line pattern found in normal thymus [30, 31 ]. The dense clusters o f epithelial cells were intermeshed with thin fibers and the lymphoid follicles were located within

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Fig. 2. Frozen section of hyperplastic MG thymus: the connective fibers visualized by monoclonal antifibronectin antibody are abnormally spread out (a) around the B cell area of the germinal center stained by a goat antihuman IgM antibody (b). • 312. this connective framework (fig. 2) in keratinnegative areas. The fibers were clearly seen around follicles having a disposition similar to that found in lymph nodes. The core constituted by dense deposits stained with antiIgM but not with anti-IgD antiserum was surrounded by a mantle of IgM+ IgD+ ceils. A T cell area (mostly CD4+ and partially CD8+) could be clearly visualized around the B cell zone (not shown). The major abnormalities in thymic hyperplasia resemble those observed in the thyroid gland in autoimmune thyroiditis [32]. Indeed, germinal centers are clearly developed in a framework of fibers often intermeshed with the thymic septae. These data suggest that the germinal centers may arise from extrathymic or preexisting vascular B cells that proliferate, leading to lymphoid organization and expansion of perivascular spaces, thus disrupting the basement membrane. Germinal center formation in peripheral lymph nodes occurs in response to the local trapping of antigen [33]. One could raise the possibility that the presence of a similar histological structure in MG thymus is the result of a response to locally trapped

antigen. Indeed, the autoantigen involved in the pathogenesis of MG is the AChR which is likely to be present in the thymus as suggested by several groups [16, 17]. The presence of germinal centers in MG thymus suggests that this might be a site of specific antibody production. Several groups [9, 10, 34] found spontaneous anti-AChR antibody production by thymic cells from MG patients but not for controls. Willcox et al. [35] found considerably greater antiAChR production when the thymic cells were dispersed with proteolytic enzymes than by mechanical ways, suggesting that enzyme treatment lead to an improved yield of germinal center cells, antigen-presenting cells and feeder cells. Spontaneous antiAChR production by thymic cells implies the local availability of antigen as already suggested [ 16, 17].

Activation Status of Lymphocytes in MG Several reports indicate that resting lymphocytes do not respond to IL2 alone, that is, without additional external stimuli Ireviewed in 36]. This is based on the fact that resting lymphocytes generate and express re-

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193

Table I. Thymocyte proliferation to r-IL2 r-lL2 concentration, U/ml

Controls, n = 17

MG patients, n = 24

12

25

50

22,934 _.+3,492

27,147 __2,793

33,355 +__3,385

p < 0.02

p < 0.001

p < 0.001

40,326 _ 4,988

59.154 + 6.545

69,395 ~-7,556

The proliferation of thymocytes to 6-day treatment with r-lL2 is expressed as Acpm (cpm with r-lL2 cpm without r-IL2). Significant differences are observed between MG and controls at 3 different r-lL2 doses.

ceptors to IL2 as a consequence of antigenic stimulation. In several pathological circumstances, alterations of IL2 receptor expression, IL2 production and proliferation to IL2 have been described. In systemic lupus erythematosus, an impaired expression of high-affinity IL2 receptor on activated lymphocytes has been shown [37] while a normal production of and responsiveness to IL2 have been noted by other authors [38]. IL2 defects have also been shown in multiple sclerosis [39-41] and rheumatoid arthritis [42] diseases. In this report, we proposed to test the ability o f freshly isolated PBL and thymocytes f r o m M G patients to respond to r-IL2 without previous stimulation. The optimal conditions for IL2 response were 6-day treatment with 20-50 U/ml r-IL2. In the thymus, the proliferative response was significantly higher in M G patients (n = 24) c o m p a r e d to controls (n -= 17) (table I; p < 0.02 for the 12 U/ml IL2 concentration and p < 0.001 for the 25 and 50 U/ml IL2 doses). However, differences in the magnitude o f the response among experiments were observed in MG, as well as in controls,

probably due to the influence o f individual i m m u n e status at the time of the study. In peripheral blood, 124 patients and 46 controls were studied, and correlations with clinical parameters were analyzed. Results are summarized in table II. Patients with high reactivity to r-IL2 were essentially those studied before t h y m e c t o m y presenting a IIB f o r m o f the disease and a significant antiA C h R Ab titer ( > 10 mS1). No correlation was observed between r-IL2 response and sex, thymus histology, T4/T8 ratio and corticosteroid treatment. The response to r-IL2 o f PBL or thymocytes was dose-dependent and could be inhibited by a blocking anti-IL2 receptor antibody (anti-TAC) which supports the specificity of the response to r-IL2 and proved that the proliferation observed is mediated by the binding of IL2 to its receptor. The high proliferative response of M G lymphocytes was correlated to an increased TAC+ cell n u m b e r evaluated by immunofluorescence. Double labeling experiments revealed that in the thymus. TAC+ cells are T cells, totally CD2+ and C D 3 7 - , C D I mostly CD3+ and partially DR+. In the

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Table 1I. Correlations between clinical data and PBL proliferation to r-IL2

Controls MG patients

Patients, n

cpm ~-SEM

p value versus controls

46 124

15,408__. 1,345 23,265 _+1,822

< 0.02

Anti-AChR titer, nM

< 0.5 0.5-10 > 10

15 4I 54

20,830+4,819 17,617+2,3t7 25,653___3,091

NS NS <0.005

Thymus histology

hyperplasia involuted thymoma

47 47 30

22,960 -~2,950 21,341 +_2,219 26,756 + 4,869

< 0.03 < 0.03 < 0.01

Disease severity

IIA IIB

50 49

20,036 • 2,495 24,538 + 2,974

NS < 0.008

< 1 > I

30 23 71

29,564 _+4,279 24,633_+ 3,548 20,160_+2,305

< 0.001 < 0.005 NS

Before thymectomy After thymectomy

The proliferation of PBL to 6-day treatment with 20 U/ml r-IL2 is expressed as Acpm.

blood, occasionally few TAC+ cells were found CD37+. However, when T and B cell populations from PBL were separated by tosetting method, the proliferative response was mainly found in the T cell fraction, indicating that the participation of B ceils to proliferation to r-IL2 is marginal. Hyperactivity is mainly found in M G thymus or in PBL o f patients before thymectomy. Thus, these hyperactive cells could originate from the thymus. After thymectomy, these ceils could decrease in the periphery, thus explaining the lower responses to r-IL2 observed 1 year after surgery. The role o f such cells in the pathogenesis o f M G is unclear but they may be involved in direct T cell mechanisms. Indeed, while t h y m e c t o m y leads to clinically favorable effects, the antiAChR antibody titers do not decrease signif-

icantly after surgery [29], indicating that other mechanisms are probably implicated in M G pathogenesis. The disappearance of hyperactivity to r-IL2 in PBL of most M G patients, associated with general clinical imp r o v e m e n t after thymectomy, supports this hypothesis. Although these lymphocytes could represent autoreactive cells involved in the pathogenesis o f MG, it should be pointed out that phenotypic signs of activated T cells (namely IL2 receptor expression) could not be detected on freshly extracted ceils. Indeed, the percentage of TAC-positive cells is similar in M G and controls before culture with r-IL2 (5% o f TAC+ cells at m a x i m a in M G or controls). T o explain the apparent contradiction between a high proliferative response to rIL2 and the low n u m b e r o f TAC-positive

Cellular Aspects of Myasthenia gravis ceils before r-IL2 treatment, several nonmutually exclusive theories can be suggested: (1) preactivated ceils from MG patients may bear IL2 receptors in low density (therefore undetectable by anti-TAC binding). (2) M G lymphocytes may have a higher ability to express high affinity IL2 receptors which are known to be involved in the interaction with IL2 and in proliferative response. Therefore a different regulation of IL2 receptor expression could occur in MG, as has been described in multiple sclerosis [39]. (3) MG lymphocytes could have receptor dysfunction related to the expression of an abnormal IL2-receptor molecule, as has been shown in T cell leukemia [43]. Establishing the validity of these nonmutually exclusive theories requires further investigation.

Proliferative Response to Synthetic A ChR Peptides of Peripheral Blood L ymphocytes Synthetic peptides and their antibodies were shown to be very useful for mapping the cholinergic binding site on AChR, preparation of species-specific anti-AChR antibodies, identification of highly immunogenie regions in the receptor and analysis and localization of phosphorylation sites in AChR [44]. The determination of myasthenogenic sites has also been reported [45]. This approach allowed to define B and T cell epitopes. Among B epitopes, MIR was found to be restricted to a region between residues 37 and 85 by immunoblot technique. Furthermore, several anti-MIR monoclonal antibodies previously produced by Tzartos and Lindstrom [46] bound preferentially to a peptide corresponding to residues 65-78 [reviewed in 47]. It is well accepted that B cell epitopes are different from T cell sites [48]. Several groups have tried to

195

define the T cell recognition sites on the AChR. Human T cell lines were isolated by selection with native torpedo AChR and it was shown that immunodominant T cell recognition sites lie on the same subunit as the MIR [reviewed in 47]. In this study, we investigated the PBL proliferation of 34 MG patients and 17 normal donors to synthetic peptides corresponding to selected domains of human or torpedo AChR. Five torpedo and 3 human peptides as well as an unrelated control peptide were used. Table III summarizes the results which could be correlated to biochemical and immunogenic characteristics of the peptides. Several points should be underlined: (1) The human peptide 169-181 [21] induces a significant proliferative response in 8 out of 34 MG patients. The corresponding torpedo peptide is positive only in 2 cases. (2) The peptide 185-196 [22] which includes a part of the ct-bungarotoxin binding site is negative whether it is from the human or the torpedo sequence. (3) The peptide 351-368 from the human sequence induces a positive response in 5 cases out of 34. With the torpedo equivalent, only 2 cases responded. (4) The torpedo peptides 330-340 and 394409 induce a positive response in 3 cases for each of the two peptides, but only in patients with a positive response to human 169-181 or human 351-368 peptides. Finally, correlations with clinical data revealed that the patients responding to peptides generally present a IIB grade severity and a high anti-AChR antibody titer. This approach using synthetic peptides demonstrates that several epitopes of AChR induce cellular proliferation of MG PBL. It is interesting to note that human peptides gave better responses than the corresponding

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Table III. Proliferative response to synthetic AChR peptides

Amino acid

Species

position

length

169-181

13 13

185-196

Positive responses

Peptide characteristics

MG patients

controls

human torpedo

8/34 2/3 t

0/17 0/16

near the a-Bgt binding site; extracellular

12 13

human torpedo

1/34 0/34

1/17 0/17

a part of the a-Bgt binding site [21]; extraceUular

351-368 351-368

18 18

human torpedo

5/34 2/34

0/17 0/17

highly immunogeneic region [22]; cytoplasmic

330-340

lt

torpedo

3/34

1/ 17

cytoplasmic

394-409

16

torpedo

3/34

0/17

Control peptide

-

-

2/34

1/17

169-I 81 185-196

Results are expressed as ratio of positive responders to the entire group of patients.

torpedo peptide and that the patients who responded to torpedo peptide also responded to the h u m a n peptide, suggesting a cross-reactivity between h u m a n and torpedo peptides as has been previously shown [21 ]. The proliferative response to some cytoplasmic sites o f AChR should be underlined. One m a y hypothesize that cytoplasmic sites o f A C h R could be accessible after damage o f A C h R by several possible mechanisms already described [18, 19]. Furthermore, the response to the 351-368 peptide is well correlated with previous studies by Souroujon et al. [23], who have shown that this peptide is highly immunogenic, and with those of Lindstrom [49], who obtained similar results by immunizing animals with denatured AChR. Finally, the demonstration of hyperactive T cells (responding to r-IL2 without Ag stim-

ulation) and o f Ag-specific autoreactive T cells as well as the important morphological and cellular abnormalities o f the thymus gland underline the hyperactivity o f the Tcell-mediated i m m u n i t y and indicate that cellular mechanisms are involved in the pathogenesis o f MG. Whether this involvement is an early or a late event in the autoimm u n e manifestation has to be investigated.

Smnmary Several cellular aspects were investigated in a large series of patients with MG. First, non-Ag-specific proliferation was tested by measuring the response to r-lL2. Thymocytes from most MG patients showed hyperactivity to r-lL2. Peripheral blood lymphocytes (PBL) from some patients also showed a high response to r-IL2. These responding patients were generally those tested before th.vmectomy, presenting a high anti-AChR Ab titer and a severe form

Cellular Aspects of Myasthenia gravis

of the disease. Second, Ag-specific proliferation of MG PBL was assayed using 8 synthetic peptides corresponding to selected domains of torpedo or human AChR. Only 2 peptides gave a positive response in a significant number of patients, essentially in those presenting high anti-AChR Ab titer. The first is located near the ct-bungarotoxin binding site and the second is in a cytoplasmic domain, according to roodels predicting the AChR transmembrane orientation. The positive results were essentially obtained with the human peptides; the corresponding torpedo peptides were positive in very few patients. Both human and torpedo peptides which include a part of the ct-bungarotoxin binding site were negative. Finally, although morphological abnormalities were clearly visible in thymic hyperplasia, no correlation could be established between the thymus type and the cellular proliferation either to r-IL2, or to the peptides. Overall, our data indicate that cell-dependent mechanisms participate in the pathogenesis of MG, but the level of their involvement deserves further investigation.

References 1 Miller, K.B.; Schwartz, R.S.: Autoimmunity and suppressor T lymphocytes. Adv. internal Med. 27.' 281-313 (1982). 2 Sch6nfeld, Y.; Schwartz, R.S.: Immunologic and genetic factors in autoimmune diseases. New Engl. J. Med. 311:1019-1029 (1984). 3 Bach, J.F.; Bach, M.A.; Carnaud, C.; Dardenne, M.; Monier, J.C.: Thymic hormone and autoimmunity; in Talal, Autoimmunity: genetic, immunologic, virologic and clinical aspects, pp. 207230 (Academic Press, New York 1977). 4 Boitard, C.; Bach, J.F.: Cell-mediated versus humoral immunity in autoimmune diseases and their pharmacologic control with particular reference to type I diabetes mellitus; in Cruse, Lewis, Concepts in immunopathology: immunoregulation and autoimmunity, pp. 193-224 (Karger, Basel 1986). 5 Castelman, B.: The pathology of the thymus gland in myasthenia gravis. Ann. N,Y. Acad. Sci. 135: 496-505 (1966). 6 Levine, G.D.: Pathology of the thymus in myasthenia gravis. Current concepts; in Dau, Plasmapheresis and the immunobiology of myasthenia

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gravis, pp. 113-123 (Houghton Mifflin, Boston 1979). 7 Papatestas, A.E.: Genkins, G.; Horowitz, S.H.; Kornfeld, P.: Thymectomy in myasthenia gravis. Pathologic, clinical and electrophysiologic correlations. Ann. N.Y. Acad. Sci. 2 7 4 : 5 5 5 - 5 7 3 (1976). 8 Le Brigand, H.; Levasseur, P.; Miranda, A.R.; Gaud, C.; Wojakowski, I.: R6sultats du traitement chirurgical de la myasth6nie (248 cas de thymectomies). Ann. Chit. 34:169-172 (1980). 9 Vincent, A.; Scadding, G.K.; Thomas, H.C.; Newsore-Davis, J.: In vitro synthesis of anti-acetylcholine receptor antibody by thymic lymphocytes in myasthenia gravis. Lancet i: 305-307 (1978). I0 Seadding, G,K.; Vincent, A.; Newsom-Davis, J.; Henry, K.: Acetylchotine receptor antibody synthesis by thymic lymphocytes. Correlation with thyrnic histology. Neurology 31:935-938 (1981). 11 Bach, J.F.; Dardenne, M.; Papiernik, M.; Barrois, A.; Levasseur, P.; Le Brigand, H.: Evidence for a serum factor produced by the human thymus. Lancet i: 1056-1058 (1972). 12 Goldstein, G.; Schlesinger, D.G.: Thymopoietin and myasthenia gravis: neostigmine-responsive neuromuscular block produced in mice by a synthetic peptide fragment of thymopoietin. Lancet ii: 256-259 (1975). 13 Venkatasubramanian, K.; Audhya, T.: Goldstein, G.: Binding of thymopoietin to the acetylcholine receptor. Proc. natn. Acad. Sci. USA 83: 31713174 (1986). 14 Morel, E.; Vernet der Garabedian, B.; Raimond, F.; Audhya, T.K.: Goldstein, G.; Bach, J.F.: Myasthenic sera recognize the human acetylchotine receptor bound to thymopoietin. Eur. J. Immunol. 17:1109-1113 (1987). 15 Revah, H.N.A.: Mulle, C.; Pinset, C.; Audhya, T.; Goldstein, G.; Changeux, J.P.: Calcium-dependent effect of the thymic polypeptide thymopoietin on the desensitization of the nicotinic acetylcholine receptor. Proc. natn. Acad. Sci. USA 84: 3477-3481 (1987). 16 Ohshima, F.; Kondo, K.; Tsubaki, T.: Binding of iodine 125 ~t-bungarotoxin to the thymus of mice. Archs Neurol., Chicago 3 5 : 3 1 - 3 2 (1978). 17 Raimond, F.: Morel, E.; Bach, J.F.: Evidence for the presence of immunoreactive acetylcholine receptors on human thymus cells. J. Neuroimmunol. 6:31-40 (1984).

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Berrih-Aknin/Cohen-Kaminsky/Neumann/Safar/Eymard/Gaud/Levasseur/Fuchs/Bach

18 Drachman, D.B.; Adams, R.N.; Josifek, L.F.; Pestronk, A.; Stanley, E.F.: Antibody-mediated mechanisms of ACh receptor loss in myasthenia gravis: clinical relevance. Ann. N.Y. Acad. Sci. 377:175-187 (1981). 19 Engel, A.G.; Sahashi, K.; Fumigalli, G.: The immunopathology of acquired myasthenia gravis. Ann. N.Y. Acad. Sci. 377:158-174 (1981). 20 Levinson, A.I.; Lisak, R.P.; Sweiman, B.; Kornstein, M.: Phenotypic and functional analysis of lymphoeytes in myasthenia gravis. Springer Semin. Immunopathol. 8:209-233 (1985). 21 Fuchs, S.; Neumann, D.; Safran, A.: What can we learn about the acetylcholine receptor from synthetic peptides?; in Maelicke, NATO ASI series, vol. H3: Nicotinic acetylcholine receptor, pp. 4959 (Springer, Berlin 1986). 22 Neumann, D.; Barchan, D.; Fridkin, M.; Fuchs, S.: Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor a-subunit. Proc. natn. Acad. Sci. USA 83: 92509253 (1986). 23 Souroujon, M.C.; Neumann, D.; Pizzighella, S.; Safran, A.; Fuchs, S.: Localization of a highly immunogenic region on the acetylcholine receptor a-subunit. Biochem. biophys. Res. Commun. 135: 82-89 (1986). 24 Levine, G.D.; Rosai, J.: Thymic hyperplasia and neoplasia. A review of current concepts. Human Pathol. 9.' 495-515 (1978). 25 MacKay, I.R.; De Gail, P.: Thymic 'germinal centers' and plasma cells in systemic lupus erythematosus. Lancet ii: 667-668 (1963). 26 McEacherns, D.; Darnell, J.L.: Relationship of hyperthyroidism to myasthenia gravis. J. clin. Endocr. Metab. 8:842-847 (1948). 27 Buckingham, J.M.; Howard, F.M.; Bernatz, P.E.; Payne, W.S.; Harrison, E.G.; O'Brien, P.C.; Weiland, L.H.: The value of thymectomy in myasthenia gravis. Mayo Clin. Proc. 184:453-457 (1976). 28 Genkins, G.; Papatestas, A.A.; Horowitz, S.H.; Kornfeld, P.: Studies in myasthenia gravis: early thymectomy: electrophysiologie and pathologic correlations. Am. J. Med. 58:517-524 (1975). 29 Berrih, S.; Morel, E.; Gaud, C.; Raimond, F.; Le Brigand, H.; Bach, J.F.: AnIi-AChR antibodies, thymic histology and T cell subses in myasthenia gravis. Neurology 34:66-71 (1984).

30 Savino, W.; Berrih, S.: Thymic extracellular matrix in myasthenia gravis. Lancet 8396:45-46 (1984). 31 Berrih-Aknin, S.; Morel, E.; Raimond, F.; Safar, D.; Gaud, C.; Binet, J.P.; Levasseur, P.; Bach, J.F.: The role of the thymus in myasthenia gravis, lmmunohistological and immunological studies in 115 cases. Ann. N.Y. Acad. Sci. 505:50-70 (1987). 32 Wick, G.; Mrst, J.; Schauenstein, IC; K.r6mer, G.; Dietrich, H.; Ziemiecki, A.; F~issler, R.; Schwartz, S.; Neu, N.; Hala, IC: Spontaneous autoimmune thyroidis. A bird's eye view. Immunol. Today 6: 359-364 (1985). 33 Klaus, G.G.B.; Humphrey, J.H.; Kunkl, A.; Dongworth, D.W.: The follicular dentritic cell: its role in antigen presentation in the generation of immunological memory. Immunol. Rev. 53:3-28 (1980). 34 Safar, D.; Berrih-Aknin, S.; Morel, E.: In vitro antiacetylcholine receptor antibody synthesis by myasthenia gravis patient lymphoeytes: correlations with thymic histology and thymic epithelial cell interactions. J. clin. lmmunol. 7." 225-234 (1987). 35 Willcox, H.N.A.; Newsom-Davis, J.; Calder, L.R.: Greatly increased autoantibody production in myasthenia gravis by thymocyte suspensions prepared with proteolytic enzymes. Clin. exp. Immunol. 54:378-383 (1983). 36 Diamantstein, T.; Osawa, H.: The interleukin-2 receptor, its physiology and a new approach to a selective immunosuppressive therapy by anti-interleukin-2 receptor monoclonal antibodies. Immunol. Rev. 92:5-27 (1986). 37 lshida, H.; Kumagai, S.; Umehara, H.; Sano, H.; Tagaya, Y.; Yodoi, J.; Imura, H.: Impaired expression of high affinity interleukin 2 receptor on activated lymphocytes from patients with systemic lupus erythematosus. J. [mmun. 139." 1070-1074 (1987). 38 Draeger, A.M.; Heleen, A.J.G.S.; van den Brink, H.G.; Aarden, L.A.: T cell function in systemic lupus erythematosus: normal production of and responsiveness to interleukin 2. a i m exp. Immunol. 64:80-87 (1986). 39 De Freitas, E.C.; Sandberg-WoIlheim, M.; Schonely, K.; Boufal, M.; Koprowsky, H.: Regulation of interleukin 2 receptors on T cells from multiple sclerosis patients. Proc. natn. Acad. Sci. USA 83:2637-2641 (1986).

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Cellular Aspects of Myasthenia gravis

40 Braakman, E.; van Tunen. A.; Meager, A.; Lucas, C.J.: Natural cytotoxic activity in multiple sclerosis patients: defects in II,-2 interferon y regulatory circuits. Clin. exp. Immunol. 66: 285-294 (1986). 41 Hofman, F.M.; yon Hanwehr, R.J.; Dinarello, C.A.; Hinton, S.B.; Merril, J.E.: Immunoregulatory molecules and IL2 receptors identified in multiple sclerosis brain. J. Immun. 136: 32393245 (1986). 42 Lemm, G.; Warnatz, H.: Evidence for enhanced interleukin 2 (IL2) secretion and IL2 receptor presentation by synovial fluid lymphocytes in rheumatoid arthritis. Clin. exp. Immunol. 6 4 : 7 1 - 7 9 (1986). 43 Yodoi, J.; Uchiyama, T.: IL2 receptor dysfunction and adult T cell leukemia. Immunol. Rev. 92: 135-156 (1986). 44 Fuchs, S.; Neumann, D.; Safran, A.; Souroujon, M.: Barchan, D.; Fridkin, H.; Gershoni, J.M.; Mantegazza, R.; Pizziehella, S.: Synthetic peptides and their antibodies in the analysis of the acetylcholine receptor. Ann. N.Y. Acad. Sci. 505: 256-270 (1987). 45 Lennon, V.A.; Griesmann, G.A.; McCormick, D.J.; Huang, Z.X.; Feng, H.; Lambert, E.H.: Definition of myasthenogenic sites of the human acetylchotine receptor using synthetic peptides. Ann. N.Y. Acad. Sci. 505:439-449 (I987).

46 Tzartos, S..I.: Lindstrom, J.: Monoclonal antibodies used to probe acetyicholine receptor structure: localization of the main immunogenicregion and detection of similarities between subunits. Proc. nam. Acad. Sci. USA 77:755-759 (1980). 47 Harcourt, G.; Jermy, A.: Mapping the autoimmunizing epitopes on acetylcholine receptors. Immunol. Today 8:319-321 (1987). 48 Finnegan, A.; Berzofsky, J.A.: Antigen recognition by T cells. Immunol. Today 7.' 317 (1986). 49 Lindstrom, J.: Probing nicotinic acetylcholine receptors with monoclonal antibodies. Trends Neurosci. 9:401-407 (1986).

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