J Neurol (1992) 239 : 82-88 dournalof

Neurology © Springer-Verlag 1992

Thymus cells in myasthenia gravis: a two-colour flow cytometric analysis of lymphocytes in the thymus and thymoma

Yoshitaka Fujii, Masanobu Hayakawa, and Kazuya Nakahara

Immunology Laboratory, First Department of Surgery, Osaka University Medical School, 1-1-50 Fukushima, Osaka, 553 Japan

Received December 10, 1990 / Received in revised form April 2, 1991 / Accepted April 9, 1991

Summary. The thymus in patients with myasthenia gravis has germinal centres and the thymic lymphocytes spon- taneously produce anti-acetylcholine receptor antibody in vitro. This suggests indirectly that the myasthenic thymus contains mature T and B cells that are autoreac- tive. We report a two colour flow cytometric analysis of the surface antigen phenotype of lymphocytes in the thymus and in thymoma in 24 patients and in 14 normal thymuses. The results show a decline in the proportion of double positive immature thymocytes with age. This process seems to be partially disrupted in patients with myasthenia gravis. In addition titres of anti-acetylcholine receptor antibody were positively correlated with the proportion of mature single positive T cells and non-T DR positive cells.

Key words: Myasthenia gravis - Thymus - Flow cyto- metry - Anti-acetylcholine receptor antibody

Introduction

Myasthenia gravis (MG) is an autoimmune disease medi- ated by antibodies to the acetylcholine receptor (AChR) of skeletal muscle [4, 5, 17, 32]. Immunosuppression by corticosteroids is effective in controlling the disease. Symptoms improve after the anti-AChR antibodies have been removed from the blood by plasmapheresis [3]. Also effective is surgical removal of the thymus, which is usually followed by a decrease in the serum level of the anti-AChR antibody [13, 21, 33]. In 10.5-16.3% of MG patients (mean 11.8%, of 2433 patients compiled from the literature), a thymoma accompanies the disease and is an absolute indication for thymectomy [22].

Although widely applied, the effectiveness of thymec- tomy is not clearly understood. Scadding et al. and others [10, 19, 24] have reported spontaneous production of anti-AChR antibody by thymic lymphocytes in MG pa- tients. This is direct evidence that the thymus is involved

Offprint requests to: Y. Fujii

in the production of anti-AChR antibody. However, it was later shown that lymphocytes from the bone mar- row, lymph node, and peripheral blood also produced this antibody [11]. This agrees with most physicians' ex- perience that antibody titres do not decrease rapidly after thymectomy, i.e. antibody production continues after thymectomy [13, 33]. However, when the amount of AChR antibodies is compared with the total IgG pro- duced in the cultures of lymphocytes from the thymus, bone marrow, peripheral blood and lymph nodes, thymus had about 10 times a higher ratio of AChR antibody/ total IgG than other lymphoid organs. Thus the produc- tion of antibodies in the thymus is more AChR-specific than in other lymphoid organs, suggesting that the thymus acts not only as an antibody-producing organ but as a site where lymphocytes are sensitized against AChR [111.

Thymic abnormalities in an autoimmune disease such as MG are interesting because the thymus is an organ where developing T cells acquire the unique properties of self-MHC restriction and self-tolerance [27, 28, 30]. The normal thymus selects T cells that are not autoreac- tive. The frequent association of thymic abnormalities in the MG thymus (germinal centres and thymomas) and the beneficial effect of thymectomy suggest that these abnormalities may result from failure of the MG thymus to render T cells self-tolerant. Thus, it is of interest to know the phenotypes of the thymic lymphocytes in MG; this may reveal the consequences of the abnormal func- tion of the MG thymus.

Thymic lymphocytes in MG have been studied using a variety of histological and immunological methods to identify different subsets of thymic lymphocytes [1, 6, 18, 26, 31]. Some studies report an increased number of B lymphocytes in the MG thymus when compared with the normal thymus. The number of B cells was corre- lated with the extent of germinal centre formation and the rate of anti-AChR antibody production by the thymic lymphocytes [18]. Most recently, dual-colour immuno- fluorescence has revealed an increased number of ma- ture T cells and B cells in MG thymus when compared with control thymus [6].

In th is r e p o r t w e u s e d t w o - c o l o u r f l o w c y t o m e t r i c ana lys is o f t h y m u s cel ls in 24 M G p a t i e n t s to i n v e s t i g a t e w h e t h e r t h e f u n c t i o n a l a b n o r m a l i t i e s a r e r e f l e c t e d by s u r f a c e p h e n o t y p e s o f l y m p h o c y t e s in t h e t h y m u s .

Patients and m e t h o d s

The patients are listed in Table 1 according to their age at thymec- tomy 12-51 years, mean 30.8 + 12.1). All the patients had weak- ness of skeletal muscle which worsened after exercise and im- proved after anticholinesterase administration and showed decre- mental response to repetitive nerve stimulation on electromyog- raphy. All except patients 2 and 5 had elevated serum anti-AChR antibodies as measured by a radioimmunoassay [16]. Normal serum samples have less than 0.3nM anti-AChR antibody. Pa- tients 3 and 13 were on low-dose (10-15 rag, alternate days) pred- nisone at the time of operation. The other patients did not receive any immunosuppressive agents. Patient 15 had associated rheuma- toid arthritis and Sjogren's syndrome.

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Thymus histology was assessed on haematoxylin and eosin stained sections from four different parts of the thymus. The ex- tent of germinal centre formation was scored as previously de- scribed [22] with a modification: grade 0, normal or involuted; grade 1, no germinal centre, some lymphocyte accumulation in the distended medulla; grade 2, one germinal centre present in at least one section; grade 3, two to four germinal centres in at least one section; grade 4, five or more germinal centres in at least one sec- tion. The amount of thymic tissue in each section differed, so the grading was not strictly quantitative. Patients 17-24 had thymomas which showed mixed lympho-epithelial morphology, except pa- tient 17 whose thymoma was predominantly epithelial. In the pa- tients with thymoma, the thymoma itself and the apparently non- tumorous thymic tissue apart from the thymoma were analysed separately. Because there was not enough uninvolved thymus in some thymoma patients germinal centre formation was divided into either involuted or hyperplastic according to the absence or presence of at least one germinal centre.

Fourteen normal thymus samples were obtained from patients without neuromuscular or immunological disorder who underwent cardiac or mediastinal surgery. The age of these patients varied

Table 1. Patient characteristics and the thymus cell phenotype. MG, Myasthemia gravis; GC, germinal centre score; grade 0, nor- mal or involution; grade 1, no germinal centre with some lympho- cyte accumulation; grade 2, 1 germinal centre in at least 1 section;

grade 3, 2-4 germinal centres in 1 section; grade 4, 5 or more ger- minal centres in 1 section (see Patients and methods); I, involu- tion; H, hyperplasia in the thymus accompanying the thymoma; ND, not determined; SD, standard deviation

Patient Age (years)/ AChRAB GC sex (nM)

Thymic lymphocytes (%)

C D 4 + 8 + C D 4 + 8 - C D 4 - 8 + C D 4 5 R A + 3 + D R + C D 3 -

MG without thyrnoma

1 12 / F 6 4 73.3 11.5 10 4.3 7.5

2 1 3 / F 0.16 3 87.3 5.1 6.3 1.1 0.9

3 15 / F 114 2 11.3 38.6 23.3 40.6 5.6

4 19 / F 0.4 3 74.2 15.7 6.5 4.9 0.9

5 20 / F 0.2 3 62.3 19.9 8.7 7.8 2.6

6 23 / F 18.2 4 18.7 41.5 14.7 26.5 10.5

7 32 / M 32 4 63.1 19 10.9 9.7 2.7

8 35 / F 169 4 39.4 30.2 11.8 13.6 5.9

9 36 / F 158 3 26.6 28.5 7.1 ND 4.6

10 36 / F 72.2 4 10.6 46.6 12.9 20.2 10.2

11 37 / F 4.4 3 54.7 24.4 6.2 13.2 7.2

12 37 / F 198 4 23.7 41 11.6 ND ND

13 38 / F 1.1 3 62.4 8.2 4.1 ND 1.8

14 45 / F 29.9 4 60.7 14.2 11.1 8.5 ND

15 45 / F 539 4 1.7 38.3 13.6 13.5 22.2

16 51 / M 10.3 0 32.1 29.1 8.4 17.4 5.5

Mean 30.8 84.6 43.9 25.7 10.5 13.9 6.3

SD (12.2) (139) (26.5) (13.0) (4.6) (10.6) (5.5)

MG with thymoma (uninvolved thymus adjacent to thymoma)

17 26 / M 4.7 I 32.3 18.4 21.1 6.2 3.0

18 38 / M 77 H 37.8 26.8 7.1 6.3 5.0

19 39 / M 27 H 37.9 18.6 13.3 12.1 6.9

20 3 9 / M 8.5 H 57.1 13.1 9.5 7.3 2.7

21 45 / F 3.7 H 37.3 27.2 20.3 30.9 9.3 22 53 / F 52 H 58.5 14.2 7.5 7.8 5.5

23 53 / M 6.1 H 23.4 25.9 12.6 6.8 10.8

24 54 / M 152 H 61.5 27 7.6 8.7 2.4

Mean 43.4 41.4 43.2 21.4 12.4 10.2 5.6 SD (9.7) (52.0) (14.0) (6.0) (5.6) (6.8) (3.0)

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Table 2. Comparison of the thymus (or thymoma) cell phenotype in the normal thymus, MG thymus or thymoma. Two thymoma patients who did not appear in Table 1 (aged 37 and 61 with anti-AChR antibody titres of 10 and 17.2 nM respec- tively) were included in this table. The thymuses of these patients were involuted and there were not enough cells for FACS analysis. Figures in parentheses are the number of thymuses or thymomas analysed. Data are presented as mean percentage, standard deviation. * Significantly different from normal, P < 0.05; ** Significantly different from normal, P < 0.001

Phenotype

CD4+8+

CD4+8-

CD4-8+

CD45RA + CD3 +

DR+CD3+

D R + C D 3 -

IL2R+CD3+

Normal (14)

MG thymus MG thymus MG thymoma (thymoma - ) (thymoma +) (10) (16) (8)

52.6, 28,1

17.0, 13.9

10.2, 5.7

5.2, 4.3

5.0, 4.9

6.9, 8.1

3.4, 2.9

43.9, 26.5 43.2, 14.0 53.1, 23.9

25.7, 13.0 21.4, 6.0 21.4, 12.9

10.5, 4.6 12.4,5.6 12.5, 7.3

13.9, 10.6" 10.2, 6.8 7.8, 7.3

4.5, 5.7 2.0,0.6 5.7, 6.6

6.3, 5.5 5.6,3.0 3.7, 2.5

1.5, 1.3 1.9, 1.3 0.5, 0.5

from i to 61 years (mean 28.1, SD 22.5). The thymus was obtained under written consent before the operation or, in some cases, the thymus was partially resected to achieve better exposure of the heart.

Heparinized peripheral blood was taken at the time of opera- tion.

Cells

The thymus cells were prepared by mechanically teasing and pres- sing the thymus or thymoma through a stainless steel mesh. Lym- phocytes were separated using Ficoll-Hypaque centrifugation. Cells were counted and viability was checked using the trypan blue dye exclusion test. Because it is not possible to recover all the cells in the thymus, the cell number reported in the text is a rough esti- mate of the actual cell number.

Antibodies

Fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labelled monoclonal antibodies were purchased from Becton Dickinson (via Fujisawa Pharmaceuticals, Osaka).

FA CS

Lymphocytes (0.5-1 x 106) in 100gl phosphate buffered saline (PBS) were incubated with 10 gl each of a combination of FITC- and PE-labelled monoclonal antibody. FITC-labelled anti-CD8, anti- HLADR, anti-IL2R, or anti-CD45RA (Leul8) and PE-labelled anti-CD4 or anti-CD3 were used. Cells were stained with prop- idium iodide 5 gg/ml just before the analysis by Facstar (Becton Dickinson) using FACScan software. Data from 10,000 cells were collected without gating and analysed using LYSIS software after gating according to the forward/side scatter pattern to identify lym- phocytes (excluding macrophages and cell debris) and rejecting cells with propidium iodide staining (dead cells). Data are pre- sented as the percentage of cells with a particular phenotype among live lymphocytes.

Statistical analysis

The proportion of the cells with a particular phenotype was ex- pressed as mean and standard deviation within the text. Correla- tion between two variables was evaluated using simple linear cor- relation with t statistics (regression coefficient presented where ap- propriate) or Spearman's rank correlation. Antibody titre was logarithmically transformed to approximate a normal distribution. Comparisons of the means of four groups in Table 2 were clone using Kruskal-Wallis analysis of variance by ranks and a multiple comparison.

100

80

o 60 o

40

2O a

0 0 70

O O

0 0

n

10 20 30 40 50 60

Age

Fig. 1. Proportion of double positive T cells in the normal thymus is negatively correlated with age (r = 0.81; P < 0.001)

Results

Normal thymus

The resul ts of the tw o-c o lou r f low cy tome t r i c analysis of the cell surface an t igen p h e n o t y p e of the l y m p h o c y t e s in the n o r m a l thymus are s u m m a r i z e d in T a b l e 2. The pro- p o r t i o n of the C D 4 + C D 8 + (doub le pos i t ive) cells is shown in r e l a t ion to the pa t i en t ' s age in Fig. 1. T h e p ro - p o r t i o n of doub le pos i t ive T cells in the n o r m a l thymus va r i ed g rea t ly f rom 0.9 to 84 .7%. This va r i a t ion was c lear ly a func t ion o f age; t he re was a s ignif icant nega t ive co r r e l a t i on (r = - 0 . 8 , P < 0.001) b e t w e e n age and the p r o p o r t i o n of doub le pos i t ive cells (Fig. 1). A g e was no t significantly co r re l a t ed with e i ther C D 4 + or C D 8 + single pos i t ive T cells in the n o r m a l thymus .

The a g e - r e l a t e d dec rease of doub le pos i t ive cells was also r e l a t e d to the n u m b e r of cells we r e c o v e r e d f rom the thymus . The n u m b e r of l y m p h o c y t e s r e c o v e r e d f rom the n o r m a l thymus d e c r e a s e d f rom 1.2 x 109/g f rom an in- fant t hymus to less than 1 x 106/g f rom the thymus in el- de r ly pa t ien t s .

MG thymus (without thymoma)

Double positive T cells (CD4+CD8). The p r o p o r t i o n of d o u b l e pos i t ive T cells in the M G thymus also va r i ed wide ly (1 .7% in pa t i en t 15 to 87.3% in pa t i en t 2) as in

100 -

o--t 8O m

o 60

40 £1_

d 3

20 C2

%

0 • 0 1 '0 2'0 3'0 4'0 5'0 6'0

Age

Fig. 2. Proportion of double positive T cells in the myasthenia gravis (MG) thymus in relation to age (r = 0.41, NS)

85

o~ v

m

o

Cz

03 E co

rm ©

50-

4O

3 0 -

2 0 -

10-

0 0.1

i

1 10 100 1000

Serum ant i -AChR ant ibody (nM)

Fig. 4. Proportion of CD4+ single positive T cells in MG thymus is positively correlated with anti-AChR antibodies in the serum (r = 0.725; P < 0.002)

100 -

o~ 80 -

o 60-

40- _~ 2 3

20 - C3

0 , • 0.1 1 10 100 1000

Serum ant i -AChR ant ibody (riM)

Fig. 3. Proport ion of double positive T cells in the M G thymus is negatively correlated with anti-acetylcholine receptor (AChR) antibodies in the serum (r = -0.8, P < 0.001)

25

2O

15

10 =+ (q

5

0 0.1 1000

i i i

1 10 100

Serum ant i -AChR ant ibody (riM)

Fig. 5. Proportion of DR+CD3- cells in the MG thymus is posi- tively correlated with auti-AChR antibodies in the serum (r = 0.81; P < 0.001)

the normal thymus. However, unlike the normal thymus (Fig. 1), the proportion of the double positive cells var- ied among the patients of similar age (Fig. 2). Thus, the proportion of double positive cells in the MG thymus did not show significant correlation with age (Fig. 2). None of the subset frequencies correlated significantly with age in MG cases. The mean percentage of the double positive cells in the MG thymus (43.9, SD 26.5%) was not significantly different from that in the normal thymus (52.6, SD 28.1%, Table 2).

We noticed that those patients with fewer double positive cells had higher anti-AChR antibody titres than those with more double positive cells. In fact, there was a significant negative correlation between the serum anti-AChR antibody titre and the proportion of double positive cells in the thymus (r = -0.7995, P < 0.001, Fig. 3). An equally significant correlation was found when 2 patients who had steroids (patients 3 and 13) were ex- cluded (r = 0.7824, p < 0.001).

Single positive T cells ( C D 4 + C D 8 - or CD4-CD8+) . The proportion of single positive cells (the C D 4 + C D 8 - cells and C D 4 - C D 8 + cells) in the MG thymus was 36.2,

SD 16.3%. This was not significantly different from that of the normal thymus (27.2, SD 16.3%). There was a sig- nificant positive correlation between the anti-AChR anti- body titre and the proportion of single positive cells (r = 0.74, P < 0.001). The proportion of CD4 single positive cells was 25.7, SD 13.0% and that of CD8 single positive cells, 10.5, SD 4.6%. Both cell types showed significant correlation with the anti-AChR antibody titre (r = 0.725, P < 0.002, for CD4 single positive cells, shown in Fig. 4; and r = 0.59, P < 0.02 for CD8 single positive cells). The proportion of C D 4 + C D 8 - single positive cells in high- titre patients (with an anti-AChR antibody of more than 10 nM) was 32.7, SD 10.4% and significantly higher than that in the age-matched control thymus (patients of age 15 or older, 19.1, SD 16.7%).

DR + C D 3 - cells. The proportion of cells with a pheno- type of D R + C D 3 - did not differ between the normal and in the MG thymus (Table 2). These cells include B cells, macrophages, and dendritic cells in the thymus cell sus- pension. Epithelial ceils probably did not contribute very much because we found very few keratin positive cells in the cell suspension from the mechanically teased thymus

86

50-

40- co

30- + c o

(D + 20 <

E~ LO

(D

• J • J

j ~ . . . J J ~ J s

i i i

1 10 100

Serum anti-AChR antibody (nM)

0 i

0.1 1000

Fig. 6. Proportion of CD45RA+ T cells in the MG thymus is posi- tively correlated with anti-AChR antibodies in the serum (r = 0.56; P < 0.05)

tissue (unpublished observation). There was a positive correlation between the proportion of D R + C D 3 - cells in the MG thymus and the serum titre of anti-AChR antibody (Fig. 5). The MG thymus with more germinal centres had more cells of D R + C D 3 - phenotype; there was a positive correlation between the germinal centre score and the percentage of D R + C D 3 - cells (rs = 0.58, P < 0.05, Spearman's rank correlation).

CD45RA+ T cells. CD45RA, an isoform of the leuco- cyte common antigen, is expressed in a subset of T cells as well as B cells. In the peripheral blood, this subset represents a population of T cells which has not been previously activated (i.e. virgin T cells). In the thymus, this phenotype is found among single positive cells and thus serves as another marker for mature thymocytes [7]. The proportion of CD45RA+ T cells (CD45RA+CD3+ cells) also showed significant correlation with the serum anti-AChR antibody titre (Fig. 6).

IL2R+ T cells. T cells which expressed IL2R were rare in the MG thymus as well as in the normal thymus (Ta- ble 2).

Non-tumorous thymus in patients with thymoma. In 10 patients with thymoma, we analysed the lymphocytes in the thymoma and in the accompanying non-tumorous thymus separately. We could not recover enough thymus cells for FACS analysis from 2 thymoma patients and these patients were omitted from Table 1. Seven of 10 thymuses in the patients with thymoma had germinal centres. We could not find any germinal centre in the sections of the thymoma in this study.

In respect to the thymus cell phenotype, we could not find any statistical difference between the thymus with- out thymoma and the thymus accompanying a thymoma (Table 2). Being a small populatin, the proportion of double positive cells in the thymus and the serum level of anti-AChR antibody titre did not show significant corre- lation (parametric and nonparametric analysis).

Thymoma. The phenotype of the lymphocytes within the thymoma in MG patients was essentially similar to that of

the accompanying thymus (Table 2). The only significant difference was found in the proportion of IL2R in the thymoma and in MG thymus without thymoma. It should be especially noted that the percentages of CD4+CD8+ cells in the thymoma in the present cases were low (19.3- 83.1; mean 53.1, SD 23.9, Table 2). This contrasts with the thymoma cells (that were lymphocytic) in non-MG patients which consisted mainly of double positive cells (42.6-92.3; mean 78.5, SD 18.3%, [12]).

Discussion

In this paper, we report a two-colour flow cytometric analysis of thymus and thymoma cells in 24 MG patients. As the thymus underwent age-related involution, the subset phenotype in the control and MG thymus also changed (Figs. 1, 2). Thus, lymphocyte population in the thymus had an inherent variability influenced by age. Because of this variability, the comparison of the means of double positive, single positive or other cell types in the normal and MG thymus did not reveal any signifi- cant difference, although the two groups had similar age distribution (Figs. 1, 2; Table 2).

However, when the patient's anti-AChR antibody titre was taken into account, it was apparent that thymus cell subpopulation frequencies correlate with the serum anti-AChR antibody titres; there was a significant nega- tive correlation between the serum anti-AChR antibody titre and the proportion of double positive cells (Fig. 3). The proportion of single positive T cells and CD45RA+ T cells in the thymus correlated positively with the serum anti-AChR antibody level (Figs. 4, 6). In other words, the thymuses in high-titre patients tended to have a higher proportion of mature T cells with a phenotype of C D 4 + 8 - or C D 4 - 8 + , CD45RA+, CD3+. They also had more cells with the phenotype of D R + C D 3 - (Fig. 5). This population includes B cells and dendritic cells (and rare epithelial cells) in the thymus cell suspension. These mature T and B cells may include autoreactive lymphocytes because thymus cells in MG patients spon- taneously produce antibodies against AChR [10, 19, 24].

The normal thymus contains a small percentage of single positive cells in the medulla. These cells mature from the cortical double positive cells after being screened for reactivity to self-MHC products [30]. Recently, many studies have been carried out on the role of the thymus in the induction of self-tolerance. These studies used antibodies that bind T cell antigen receptors (TCRs) with reactivities to specific antigens or transgenic mice with TCRs of defined specificites. These studies were in- tended to follow the fate of autoreactive lymphocytes. They have shown that T cells which express a TCR spe- cific to the self-MHC product in the thymus are deleted during their development in the thymus [14, 15, 20, 29]. Thus, it may be reasonable to speculate that AChR or its derivatives associated with self-MHC on thymic non- lymphoid cells [8, 25], if present in sufficient amount, normally serves as an antigen to induce tolerance.

The present study shows that in the thymus of the MG patients with high anti-AChR antibody titres, there

87

were increased frequencies of single positive cells, perhaps including some with potential reactivity to self-AChR. This suggests that the abnormalit ies seen in the M G thymus (and thymoma) may be related to the function of the thymus to render T cells self-tolerant and that M G is a result of a nonspecific breakdown of self-tolerance. This is supported by a frequent association of other auto- immune diseases in M G patients [2, 9, 23]. Moreover , the increased number of single positive cells found in high-titre patients are certainly not all AChR-specific be- cause even in rats hyper immunized with foreign AChR, the frequency of AChR-reac t ive cells in limiting dilution cultures is less than 1/10000 (Y. Fujii, unpublished ob- servation).

Our data do not indicate the origin of the autoreac- tive T cells and B cells seen in the M G thymus; they may be the cells that have matured de novo in the thymus or they may be imigrant peripheral blood lymphocytes. The increased proport ion of B cells (which are supposed to mature elsewhere than the thymus) supports the latter idea. The M G thymus may have a microenvironment that favours imigration of circulating blood lymphocytes including those that are activated with self-antigens.

The present study supplements previously repor ted studies on thymic lymphocytes in M G [1, 6, 18, 26, 31] with two colour flow cytometric analysis of the M G thy- mus. Among previous reports, a recent single and dual colour analysis of the thymus cells in M G patients by Durelli et al. [6] repor ted similar results that are in gen- eral agreement with the results of the present study.

CD1 has been the most widely used marker for corti- cal thymocytes until the two-colour FACS analysis be- came available. Using three-colour FACS and cell sep- aration using magnetic beads, C D I + and C D 4 + C D 8 + populations are closely overlapping. A recent detailed analysis [26] using anti-CD1 ant ibody showed clear in- verse age correlation with the area occupied by cortex, the percentage of CDl+ce l l s , and the absolute number of CD1 + cells in M G thymus, which is in good agree- ment with the present study. However , these authors and Durelli et al. failed to detect significant correlation between the percentage of C D I + cells in the thymus and the serum ant i -AChR antibody titre. Patient selection and a yet undefined difference between CD1 and CD4/ CD8 populations may have caused this different finding.

Acknowledgement. This work was supported in part by a Grant-in- Aid from the Ministry of Culture and Education and the Ministry of Health and Welfare.

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