The effect of thymectomy on autoreactive T- and B-lymphocytes in myasthenia gravis

Journal of Neuroimmunology 74 Ž1997. 45–54

The effect of thymectomy on autoreactive T- and B-lymphocytes in myasthenia gravis ˚ Richard Ahlberg a

a,)

, Qing Yi a , Ritva Pirskanen c , Georg Matell c , Ann-Charlott Sundevall a , b ˚ Bengt Aberg , Ann Kari Lefvert a

Immunological Research Laboratory and Section of Hematology and Immunology, Department of Medicine, Karolinska Hospital, 171 76 Stockholm, Sweden b Department of Thoracic Surgery, Karolinska Hospital, 171 76 Stockholm, Sweden c Department of Neurology, Soder ¨ Hospital, 118 83 Stockholm, Sweden Received 29 May 1996; revised 1 October 1996; accepted 2 October 1996

Abstract Eleven patients with myasthenia gravis were followed for three years after thymectomy. Acetylcholine receptor-specific T-cell stimulation was found in 8r11 patients before operation as compared to 2r11 three years after thymectomy. Changes of T-cell antireceptor-reactivity were commonly paralleled by changes in disease severity. The numbers of cells secreting IL-2 upon stimulation with human acetylcholine receptor correlated with those secreting IFN-g. T-cell reactivity against a monoclonal acetylcholine receptor antibody did not decrease after thymectomy. Such reactivity could reflect a beneficial immune response counteracting anti-receptor reactivity. The frequency of autoantibody-secreting cells remained unchanged, while the serum concentration of acetylcholine receptor antibodies started to decrease one year after thymectomy. All examined thymus-cell suspensions contained autoreactive T- and B-lymphocytes. There was a preferential enrichment of autoreactive lymphocytes in the thymus in a few patients with recent onset of disease Keywords: Myasthenia gravis; Thymectomy; Thymus; T lymphocyte; Cytokines

1. Introduction Several lines of evidence indicate a crucial role of the thymus in the pathogenesis of the autoimmune disease myasthenia gravis ŽMG.. The patients display a high prevalence of thymic abnormalities; lymphofollicular hyperplastic changes are present in 60–80% of the patients, and epithelial neoplasia in 10–15% ŽCastleman, 1966.. The autoantigen in MG, the acetylcholine receptor ŽAChR. at the neuromuscular junction, is also expressed by thymic myoid cells and an AChR-like protein is expressed by thymic epithelial cells ŽEngel et al., 1977; Kirchner et al., 1988a; Marx et al., 1990; Schluep et al., 1987.. B cells secreting antibodies ŽAb. against the AChR and AChR-reactive T cells are present in the MG thymus ŽMelms et al., 1988; Scadding et al., 1981; Vincent et al., 1978..

) Corresponding author. Tel.: q46-8-7295488; fax: q46-8-317303; e-mail: [email protected]

Thymectomy usually induces improvement of muscle function and increased remission rate, especially in patients without thymoma and with short duration of disease ŽBuckingham et al., 1976; Keynes, 1949; Oosterhuis, 1983; Papatestas et al., 1987.. The mechanism responsible for this improvement is still unclear. In the present study, we examined the effect of thymectomy on the immune system by serial determinations of Tand B-cell autoreactivity. Thymus cell suspensions from eight of the eleven patients were examined in parallel.

2. Patients and methods 2.1. Patients Eleven consecutive patients with MG undergoing thymectomy were included in the study. No patient was excluded for any reason. The clinical features are summarized in Table 1. The diagnosis was confirmed by demon-

0165-5728r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 6 . 0 0 2 0 4 - 4

˚ R. Ahlberg et al.r Journal of Neuroimmunology 74 (1997) 45–54

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Table 1 Patient characteristics and clinical response to thymectomy Age Sex Pre-op Disease Disease AChR-Ab duration severity Žyears. titre Žnmolrl.

a

Thymus histology

Clinical evaluation

A 61 B 29

M F

32 0

2.5 17

IIA IIA

atrophy hyperplasia

qq 0ry

C D E F

25 24 26 68

F F M F

14 25 21 18

3 0.8 0.3 0.5

IIA IIA IIB IIB

hyperplasia hyperplasia mixed cell thymoma epithelial cell thymoma

qq qq y y

G H I J K

61 25 22 50 38

M F F F M

25 42 30 30 22

20 1.5 1 1.8 0.8

IIA IIB IIA IIA IIB

atrophy hyperplasia hyperplasia mixed cell thymoma atrophy

qq qq qq y qq

Immunosuppressive treatment and other characteristics c

type 1 diabetes mellitus seronegative MG hypothyroidism rheumatoid arthritis familial colonpolyposis

treatment with azathioprin initiated 3 months after thymectomy treatment with azathioprin and prednisone initiated 12 months after thymectomy pregnant

b

treatment with azathioprin initiated 4 months after thymectomy

a

Clinical staging before thymectomy according to the Osserman–Oosterhuis classification. 25th week of pregnancy at the time of thymectomy. c Clinical evaluation 12 and 36 months after thymectomy. ‘qq’s marked improvement, ‘0ry’s unchanged at 12 months and disease progression at 36 months,‘y’s disease progression. b

strating a typical muscular fatigability, a positive response to acetylcholinesterase inhibitors, typical findings on single fiber electromyography and repetitive nerve stimulation tests and by the presence of AChR Ab in serum in 10 out of 11 patients. Clinical stage was determined according to the Osserman–Oosterhuis classification ŽOosterhuis, 1964.. Seven of the patients were in stage IIA Žmild generalized MG. and four were in stage IIB Žsevere generalized MG. at the time of thymectomy. A clinical and immunological evaluation was done one month before and 6, 12 and 36 months after the operation. An extended thymectomy was performed through a transsternal approach by the same ˚ .. The operation involved an ‘en bloc’ excisurgeon ŽB.A. sion of the thymus together with all anterior mediastinal adipose tissue ŽMasaoka et al., 1975.. 2.2. Clinical eÕaluation The clinical condition of the patients was monitored by standardized muscle function tests developed and routinely used at the Myasthenia gravis center, Soder Hospital, ¨ Stockholm, Sweden. The test protocol can be obtained from the author ŽR.P... Fatigability of extraocular-, bulbarand upper and lower extremity muscles was tested. The examinations were performed in the morning at 8.30 by the same physicians ŽR.P. and G.M... The result of thymectomy was classified as disease progression, no change, improvement Žeither diminished need for cholinesterase inhibitors or a better clinical response to it., marked improvement Žboth diminished need for cholinesterase inhibitors and a better clinical response to it., and remission Žno cholinesterase inhibitors, no myasthenic symptoms.. The clinical state, the serum antibody

levels and the cellular responses were assessed blind of each other by separate investigators. 2.3. Preparation of cells Thymus cell suspension was prepared by mechanically teasing and pressing fresh thymic tissue against a stainless steel mesh ŽFujii et al., 1992.. Mononuclear cells were isolated using Ficoll-Hypaque ŽPharmacia, Uppsala, Sweden. density gradient centrifugation. The cells were washed twice and resuspended in RPMI 1640 medium supplemented with L-glutamine Ž4 mM., penicillin Ž100 IUrml., streptomycin Ž100 mgrml. and 10% heat-inactivated pooled human ABRhq serum. Viability was ) 90% as assessed by the Trypan blue dye-exclusion test. Peripheral blood mononuclear cells ŽPBMC. were isolated by Ficoll–Hypaque density gradient centrifugation of heparinized blood and washed twice before resuspension in RPMI 1640 medium supplemented as above. The immunological analyses were performed in ignorance of whether the cells were from patients or controls. 2.4. T-cell stimulation by human muscle AChR and by a human monoclonal receptor antibody Two antigens were used for stimulation of T cells. One antigen was human muscle AChR, affinity-purified and concentrated by the binding to a-bungarotoxin immobilized on microtiter plates as described elsewhere ŽYi et al., 1993.. The same batch of musclepreparation was used at the same predetermined dilution throughout the study. The other antigen was a human monoclonal AChR antibody ŽAChR-mAb., produced by an Epstein–Barr virus trans-

˚ R. Ahlberg et al.r Journal of Neuroimmunology 74 (1997) 45–54 Table 2 The stimulated numbers of IFN-g and IL-2 secreting cellsr10 5 PBMC induced by human muscle AChR and AChR-mAb Ž2-115-19. in healthy controls IFN-g secreting cells AChR AChR-mAb

4.0"3.2 3.8"5.0

a

IL-2 secreting cells 3.8"2.8 2.6"4.3

47

secreting cells per 10 5 mononuclear cells. Stimulated numbers of cytokine-secreting cells exceeding the mean " 2SD of the mean of 28 healthy individuals ŽYi et al., 1993; Yi et al., 1992., were considered as disease-specific T-cell responses ŽTable 2.. All assays were performed in duplicate. The coefficient of variation between duplicate values was 8.5%.

a

Mean"SD of the mean. Adopted from Yi et al. Ž1993. and Yi et al. Ž1992..

formed B-cell clone from peripheral lymphocytes from a patient with MG ŽLefvert and Holm, 1987.. This antibody was an IgG-k mAb that recognizes human muscle AChR and a recurrent idiotype expressed on immunoglobulins in 60% of patients with MG ŽLefvert, 1987; Lefvert and Holm, 1987.. It stimulates T cells from 80% of the patients to secrete IFN-g and IL-2 ŽYi et al., 1992.. AChR-mAb purity was confirmed at the gene level ŽVictor et al., 1992.. T-cell stimulation in response to these antigens was examined by enumerating IFN-g and IL-2 secreting cells using an enzyme-linked immunospot ŽELISPOT. assay ŽYi et al., 1992.. In all experiments, PBMC were cultivated in the presence of human muscle AChR or AChR-mAb Ž10 pgrml.. Cells incubated in complete medium only were used to detect cells that spontaneously secreted cytokines. Control samples consisted of a-bungarotoxin without muscle extract, muscle extract without toxin, and Con A Ž2.5 and 20 mgrml.. The number of spots derived in cultures without antigen Žspontaneous IFN-g or IL-2 secretion. was subtracted from the values derived in cultures with addition of AChR or AChR-mAb, thus giving the stimulated number of cells. This has earlier been shown to reflect the prevalence of antigen-stimulated T cells ŽYi et al., 1993.. The data are expressed as numbers of IFN-g or IL-2

2.5. Cells secreting AChR IgG Ab and IgG An ELISPOT assay, using human muscle receptor bound to a-bungarotoxin as antigen, was used for enumeration of cells secreting AChR Ab of IgG isotype ŽYi et al., 1993.. Control samples consisted of a-bungarotoxin alone and muscle extract alone. Values were given as numbers of antibody-secreting cells per 10 6 mononuclear cells. The coefficient of variation between duplicate cultures was 9%. Determination of total number of IgG-secreting cells was performed in parallel using wells coated with goat anti-human IgG as previously described ŽYi and Lefvert, 1992.. 2.6. Antibodies against the AChR The concentration of AChR Ab in serum was determined by radioimmunoassay as previously described ŽLefvert et al., 1978.. 2.7. Statistical analysis Wilcoxon signed rank test was used for statistical comparison of the results obtained before and after thymectomy in the T- and B-cell ELISPOT assays. Simple linear regression was used for analysis of correlation. The level of significance was defined as p - 0.05.

Table 3 Peripheral blood mononuclear cell response to human AChR and AChR-mAb Ž2-115-19. examined before and after thymectomy Patient

A B C D E F G H I J K a

Clinical evaluation

qq 0ry qq qq y y qq qq qq y qq

b

Response to AChR a Žstimulated No. of cells secreting IFN-grIL-2.

Response to AChR-mAb a Žstimulated No. of cells secreting IFN-grIL-2.

pre-op

post-op 12 months

post-op 36 months

pre-op

post-op 12 months

post-op 36 months

23r25 40r27 35r15 11r7 6r2 8r10 4r12 6r7 20r6 15r5 10r5

21r15 3r5 4ry 5 6r8 2r6 11r24 2r6 3r4 26r20 4r5 3r4

4r4 16r17 6r8 6r6 4r5 2r4 4r4 16r15 4r6 2r8 4r6

14r28 30r39 13r8 6r6 19r4 8r10 3r7 7r8 24r8 13r13 14r14

28r6 2r4 6r5 y1r3 6r5 5r8 6r6 5r4 19r7 10r15 7r9

12r4 6r16 10r12 3r3 4r6 4r10 4r8 11r14 20r12 4r6 0r6

Disease-specific peripheral blood mononuclear cell ŽPBMC. response Žitalics. was defined as a stimulated number of IFN-g or IL-2 secreting cells exceeding the mean " 2SD of the mean of healthy individuals ŽTable 2.. b Clinical evaluation 12 and 36 months after thymectomy. ‘qq’s marked improvement, ‘0ry’s unchanged at 12 months and disease progression at 36 months, ‘y’s disease progression.

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3. Results 3.1. Clinical response The clinical response to thymectomy was evaluated one and three years after the operation. A marked improvement of muscle function was evident in 7 of the eleven patients ŽTable 1.. All three patients with thymoma deteriorated

after thymectomy but improved after initiation of immunosuppressive treatment. In two of them ŽE and J. azathioprine treatment was initiated a few months after thymectomy and in one of them ŽF. prednisone and azathioprine treatment was initiated after 12 months. In one patient ŽB. with hyperplasia and 17 years duration of MG, the disease severity was unchanged until three years after thymectomy when disease progression was observed.

Fig. 1. Numbers of T cellsr10 5 PBMC stimulated to secrete IFN-g by incubation with ŽA. human acetylcholine receptor, or ŽB. a human monoclonal acetylcholine receptor antibody. The data are obtained after subtraction with the numbers of IFN-g secreting cells found in non-stimulated cultures examined in parallel.

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3.2. Spontaneous and Con A induced secretion of IFN-g and IL-2 The numbers of PBMC with spontaneous secretion of IFN-g and IL-2 decreased after thymectomy Ž p - 0.01. ŽFig. 3.. Before, but not after thymectomy, the numbers of cells secreting IFN-g correlated with those secreting IL-2 Ž r s 0.76, p - 0.01.. The numbers of cytokine-secreting cells in cultures exposed to a-bungarotoxin and muscle extract alone did not differ from the numbers in cultures without toxin and muscle extract. Addition of Con A Ž2.5 and 20 mgrml. to the cultures induced increased numbers of cells secreting IL-2 and IFN-g Ž p - 0.01.. The responses to Con A after thymectomy did not differ from the preoperative responses. In thymus cell suspensions, the median number of cells with spontaneous secretion of IFN-g was 16 Žrange 0–80. per 10 5 mononuclear cells and the median number of IL-2 secreting cells was 24 Žrange 0–50. per 10 5 mononuclear cells. The highest numbers were found in a patient ŽB. with hyperplasia and 17 years’ duration of disease. 3.3. T-cell response to AChR and AChR-mAb Disease-specific stimulation of T cells, determined as the stimulated numbers of cytokine-secreting cells exceeding the mean " 2SD of the mean of healthy individuals ŽTable 2., was demonstrated preoperatively in 8r11 patients in response to AChR and in 6r11 in response to AChR-mAb ŽTable 3.. Three years after thymectomy the corresponding figures were 2r11 and 4r11, respectively. The frequency of cells secreting IFN-g in response to AChR decreased after thymectomy, while the frequency of AChR-mAb-reactive T cells was not affected ŽFig. 1A,B.. The stimulated numbers of cells Žthe numbers obtained after subtraction with the numbers of cytokine-secreting cells in the non-stimulated cultures. secreting IFN-g in response to AChR correlated with those secreting IL-2, both before Ž r s 0.73, p - 0.02. as well as 12 Ž r s 0.72, p - 0.02. and 36 months after thymectomy Ž r s 0.86, p - 0.001.. The total numbers of cytokine-secreting cells Žunsubtracted number. in cultures stimulated with either AChRtoxin complex or AChR-mAb were, before as well as after thymectomy, significantly higher than the numbers obtained in the corresponding non-stimulated culture Ž p 0.01.. The highest numbers of AChR-reactive T cells before thymectomy were found in two patients ŽB and C. with thymic hyperplasia and long duration of disease. The patient C improved markedly after thymectomy and disease specific T-cell reactivity against AChR was not detectable one or three years after the operation. In the other patient ŽB. the disease severity was not influenced by thymectomy and disease specific T-cell reactivity against AChR was present three years after thymectomy. The only

Fig. 2. ŽA. Numbers of T cellsr10 5 mononuclear cells stimulated to secrete IFN-g by incubation with human acetylcholine receptor. The data are obtained after subtraction with the numbers of IFN-g-secreting cells found in non-stimulated cultures examined in parallel. ŽB. Numbers of B cellsr10 6 mononuclear cells secreting antibodies against the acetylcholine receptor. Peripheral blood was examined one month before thymectomy.

patient ŽH. with marked improvement of disease who displayed increased frequency of AChR reactive T cells after three years, also displayed increased frequency of AChR-mAb reactive T cells ŽTable 3.. The highest frequency of such idiotype-reactive T cells at the three-year follow up was found in a patient ŽI. with complete remission of disease. Mononuclear cells obtained from the thymus were compared with corresponding PBMC from 8 of the patients. The numbers of cells secreting IFN-g in response to AChR in the individual patients are shown in Fig. 2A. The only patient ŽH. in whom neither AChR- nor AChR-mAbspecific T-cell stimulation was demonstrated in peripheral blood before thymectomy, displayed the highest frequency AChR reactive thymic T cells. The stimulated number of IFN-g secreting cells was 42r10 5 Ž1r2400. mononuclear

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Fig. 3. Numbers of T cellsr10 5 PBMC secreting IFN-g in non-stimulated cultures.

cells, and the corresponding figure for IL-2 secreting cells was 18r10 5 Ž1r5600. mononuclear cells. 3.4. Antibody secreting cells in peripheral blood and thymus cell suspension AChR-Ab secreting cells were detectable in peripheral blood in 8r11 patients before thymectomy, as compared to 11r11 after 36 months ŽFig. 4.. The frequency of such cells in peripheral blood was not clearly affected by

thymectomy. AChR-Ab secreting cells were demonstrated in all 8 examined thymuses with a mean of 4 cellsr10 6 mononuclear cells ŽFig. 2B.. The numbers of AChR-Ab secreting cells did not correlate with the total numbers of IgG-secreting cells, either in peripheral blood or in the thymus. Two patients with thymus hyperplasia and disease duration less than one year ŽD and I. had no detectable AChR-Ab secreting cells in peripheral blood before thymectomy, but such cells were present in the thymus and in peripheral blood 6, 12 and 36 months after thymectomy.

Fig. 4. Numbers of B cellsr10 6 PBMC secreting antibodies against the acetylcholine receptor ŽAChR Ab..

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Fig. 5. Serum concentration of acetylcholine receptor antibody ŽAChR Ab. before and after thymectomy.

3.5. Antibodies against human AChR The serum concentrations of AChR Ab were not significantly altered during the first year after thymectomy, thereafter it decreased in all the seropositive patients ŽFig. 5.. One patient ŽB. had no detectable AChR Ab in serum at repeated examinations, but cells secreting such antibodies were present both in peripheral blood and in thymus cell suspension.

4. Discussion A clinical improvement was demonstrated in 7r11 patients; four young females with thymus hyperplasia and recent onset of disease, and three men with thymus atrophy. Three patients with thymoma deteriorated after thymectomy. This different response to thymectomy dependent on the histology of the thymus and duration of disease is entirely in accordance with previous studies ŽKeynes, 1949; Oosterhuis, 1983; Papatestas et al., 1987.. Disease-specific T-cell stimulation by AChR was found in eight patients before thymectomy. After three years it remained in one of those eight patients, one who did not improve after thymectomy. The three patients who preoperatively did not display such a response had short duration of disease Ž- 1.5 years., suggesting that AChR-reactive T cells secreting IFN-g and IL-2 could be less frequently present in peripheral blood at an early stage of disease. The effect of thymectomy on disease-specific T-and B-cell reactivity is difficult to evaluate in the three patients with thymoma since they all received immunosuppressive

treatment due to disease progression. After initiation of treatment, all three improved with an accompanying reduction in AChR-Ab titre and in the number of cells with disease-specific T-cell reactivity. One of the patients with thymoma and disease progression who was without immunosuppressive treatment until one year after thymectomy, was the only patient with increased T-cell reactivity against AChR at the one year follow up. This patient was still treated with prednisone at the three-year follow up when disease specific T-cell autoreactivity was no longer detectable. One could raise doubts about the specificity and relevance of the high T-cell response to the AChR in the patient with hyperplasia without detectable AChR Ab in serum. However, the lack of detectable AChR Ab in serum did not reflect an inability to produces such antibodies. Cells from this individual secreted AChR Ab in in vitro cultures thus confirming the presence of specific response against the AChR. The results suggest that changes in the numbers of AChR-reactive T cells often parallel major changes in muscle function. One exception was a patient ŽH. with hyperplasia and durable marked improvement after thymectomy. This person did not display disease-specific T-cell reactivity before or during the first year after thymectomy, but such reactivity was detected against both AChR and AChR-mAb at the three-year follow up. It is not clear whether the observed occurrence of T cells with reactivity against the monoclonal AChR Ab reflects the presence of a protective response counteracting the effect of AChR-reactive T-and B-cells, or whether it merely reflects the presence of an ongoing autoreactive process. The former hypothesis could explain why the patient men-

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tioned above did not deteriorate despite increased T-cell reactivity against AChR. An other observation supporting this hypothesis is a dominance of idiotype-reactive T cells as compared to AChR-reactive cells in several of the patients with improved muscle function at the three-year follow up. Such a dominance was most pronounced in the only patient ŽI. who achieved complete remission. All cultures of thymic mononuclear cells stimulated with AChR displayed higher numbers of cytokine secreting cells than corresponding non-stimulated cultures. The highest response was found in a patient with short duration of disease who lacked AChR-specific T-cell response in peripheral blood. AChR-Ab secreting B cells were demonstrated in all examined thymuses, but not in blood samples from three patients with recent onset of disease. The results indicate a possible preferential localization of autoreactive T- and B-cells in MG thymus in some patients with recent onset of disease. Such enrichment at an early stage of MG has previously been indicated by a report on increased secretion of AChR Ab and increased proliferative response to AChR in cultures of thymocytes as compared with cultures of PBMC ŽSommer et al., 1990.. Another study showed the quotient between AChR Ab and total IgG spontaneously produced in cultures of thymic lymphocytes to be about ten times higher than in cultures of bone marrow, peripheral blood, or lymph node ŽFujii et al., 1986.. This difference was most pronounced in patients with recent onset of disease. Thymic lymphocytes obtained from MG patients with thymoma produce AChR Ab, but it is rare that the lymphocytes from the thymoma itself produce these antibodies Fujii Neurology 34 1182 1984. Thus it is possible that the observed AChR-reactive cells in thymic cell cultures in our study originate from areas adjacent to the thymomas even though hyperplastic areas were not seen upon histological examinations. In the histologically atrophic MG thymuses it may exist minor changes resembling those in hyperplastic thymuses ŽEimoto et al., 1989., which could explain our finding of autoreactive cells in this tissue. Our study demonstrated that peripheral blood T-cell reactivity against human AChR decreased after thymectomy. This is in line with a previous report of reduced proliferative response to Torpedo AChR after thymectomy ŽMorgutti et al., 1979.. Such a decreased autoreactivity was probably not caused by increased endogenous steroidproduction due to surgical stress, since it progressed and lasted during the three years of follow-up. The decreased reactivity could rather be due to removal of autoreactive T cells present in the thymus, removal of the main activation site for such T cells, or both. It has been proposed that the autoimmune process of MG originates within the thymus by an aberrant confrontation of AChR-expressing myoid cells with AChR-specific autoimmune T lymphocytes ŽKirchner et al., 1988b; Wekerle and Ketelsen, 1977.. Others have suggested that AChR-specific T cells are enriched in the hyperplastic thymus after prior sensitiza-

tion elsewhere ŽSommer et al., 1990.. Both hypotheses include activation of autoreactive T cells within the thymus. The observed diminished T-cell autoreactivity in this study could be one mechanism for the therapeutic effect of thymectomy. An association between T-cell autoreactivity and disease activity is also suggested by our report on anti-CD4 mAb treatment in a patient with severe MG ˚ ŽAhlberg et al., 1994.. In this patient the marked clinical and electrophysiological improvement and the subsequent deterioration closely paralleled the autoantigen-induced Tcell stimulation. Another observed effect of thymectomy was the diminished numbers of IFN-g secreting cells in non-stimulated cultures, which is in line with a report of diminished serum levels of soluble IL-2-receptor after thymectomy ŽCohenKaminsky et al., 1992.. These data show that activated T cells are commonly present in peripheral blood and decline after thymectomy. This is not believed to be due to a general reduction of one or more of the major lymphocyte subsets since most previous studies on subset changes after thymectomy demonstrate either no changes ŽCox et al., 1986; Melms et al., 1993., or only a modest and transient decrease of the CD8 q cells ŽBerrih et al., 1981; Kawanami et al., 1990.. However there is also one report on a durable decrease of the CD4 q cells after thymectomy ŽBerrih et al., 1983.. It could instead be due to a slow elimination of a long-lived activated cell fraction generated in the thymus. Some of these cells were possibly AChR-specific. This was indicated by the gradual reduction of AChR-reactive cells, which preceded and paralleled the major fall in the number of non-stimulated IFN-g secreting cells. The ELISPOT assay, employed in this study to detect and quantify antigen-specific T cells, has previously been carefully evaluated from a methodological point of view ŽKabilan et al., 1990.. It has frequently been used to quantify T-cell response to autoantigens in patients with MG ŽSun et al., 1992; Yi et al., 1993; Yi et al., 1992. and other autoimmune disorders ŽOlsson et al., 1990; Sun et al., 1991.. In the present study the numbers of AChR-reactive cells secreting IFN-g correlated with those secreting IL-2, both before as well as in the serial examinations after thymectomy. Human CD4 q T cells can be subdivided according to the cytokine-secretion pattern. TH1 cells secrete IFN-g and IL-2 but not IL-4, TH2 cells secrete IL-4 but not IFN-g or IL-2, and TH0 cells secrete all three cytokines ŽRomagnani, 1991.. Thus, the T cells secreting IFN-g and IL-2 in response to AChR or AChR-mAb in this study could represent TH1-or TH0-like CD4 q cells. Also CD8 q cells and NK cells may have contributed to the numbers of cytokine secreting cells. NK cells secrete IFN-g, but are not directly stimulated by antigens. Since the response to the AChR was dependent on the presence of monocytesrmacrophages and MHC class II, the cells secreting IFN-g and IL-2 upon stimulation with these antigens are considered to be CD4 q T cells ŽYi et al., 1994..

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Previous studies demonstrated that the serum levels and the in vitro synthesis of AChR Ab declined after thymectomy ŽScadding et al., 1977; Vincent et al., 1983; Kuks et al., 1992.. In the long-term follow-up studies such decline correlated with clinical improvement but numerous individual exceptions were noted ŽScadding et al., 1977; Vincent et al., 1983.. In the present study the AChR-Ab concentration in serum remained stable during the first year after thymectomy. Thereafter it decreased in all patients, both in those seven who improved after thymectomy and in those three who deteriorated but were instituted on immunosuppressive treatment. This was not due to a decreased frequency of AChR-Ab secreting cells in peripheral blood. The mechanism could be the removal of an organ releasing autoantibody producing B cells into the blood. It could also be secondary to the observed reduction in T-cell autoreactivity with subsequent reduced activation of AChR-Ab secreting cells. It could be of importance that the three patients without detectable AChR-Ab secreting cells in peripheral blood before thymectomy, displayed such cells in repeated examinations of peripheral blood after thymectomy. Thymus cell suspension, examined in two of these three patients, also contained AChR-Ab secreting cells. Perhaps it reflects that autoreactive lymphocytes escaped the thymus to peripheral blood during the operation. Maybe the therapeutic effect of thymectomy could improve if the vessels supporting the thymus were ligated before the gland is mechanically agitated during the dissection. This study shows that T-cell reactivity against the AChR decreases after thymectomy and that such reactivity tends to correlate with disease severity. Our data clearly show the presence of autoreactive T- and B-lymphocytes in thymus and indicate that such cells could be preferentially localized to the thymus in a few patients with short duration of disease. This could explain the clinical observation that the highest remission rate is found in patients with duration of MG less than one year ŽBeghi et al., 1991; Papatestas et al., 1981., and it further emphasizes the importance of early thymectomy in MG. Acknowledgements The skilled technical assistance of Mr. Ricardo Giscombe, Mrs. Margareta Soderqvist, Mrs. Juta Andersson ¨ and Mrs. Wen He is gratefully acknowledged. This work was supported by grants from the Swedish Medical Research Council Ž05646., the Palle Ferb foundation, the Nanna Svartz foundation, and the foundations of the Karolinska Institute. References ˚ Ahlberg, R., Yi, Q., Pirskanen, R., Matell, G., Swerup, C., Rieber, P., Rietmuller, G., Holm, G. and Lefvert, A.K. Ž1994. Treatment of ¨

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