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Imbalances in T-cell subpopulations in myasthenia gravis
Journal of the Neurological Sciences, 1981,52:53-59 Elsevier/North-Holland Biomedical Press
53
IMBALANCES IN T-CELL SUBPOPULATIONS IN MYASTHENIA GRAVIS
HOU-CHANG CHIU l, KUE-HSIUNG HSIEH 2 and TSU-PEI HUNG ~
IDepartment of Neurology and 2pediatrics, National Taiwan University Hospital, Taipei, Taiwan 100 (Republic of China) (Received 10 December, 1980) (Revised, received 12 March, 1981) (Accepted 19 March, 1981)
SUMMARY
Distributions of peripheral blood lymphocyte subpopulations and T-cell subsets were studied in 38 patients with myasthenia gravis (MG) and 23 healthy controls. T cells were detected by rosette formation with sheep red blood cells and B cells with erythrocyte-antibody-complement (EAC) complexes. T# cells were identified by rosette formation of T cells with Ox RBC-IgM complexes, and Ty cells with Ox RBC-IgG complexes. The means of total lymphocyte count and active T cell percentage were marginally significantly lower in MG patients than in normal controls (0.05
INTRODUCTION
Myasthenia gravis (MG) has been considered as an autoimmune disease on the basis of clinical manifestations (Simpson 1966) and laboratory findings (Kott This study was supported by a grant from the National Council of Science, Republic of China ; grant No. NSC-70B-0412-02(04). Address for correspondence: Dr. Hou-Chang Chiu, Department of Neurology, National Taiwan University Hospital, 1, Chang-Te Street, Taipei, Taiwan 100, Republic of China. 0022-510X/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
54 et al. 1973; Almon et al. 1974; Lindstrom et al. 1976). Abnormal lymphocyte functions in MG have been reported by several investigators (Kawanami et al. 1979; Chiu et al. 1980). Although no significant difference in peripheral blood lymphocyte subpopulations between MG patients and control subjects was found (Shirai et al. 1976; Chee et al. 1978), the normal counts could not exclude the possibility of quantitative and/or qualitative abnormalities of T cell subpopulations in MG patients. If an autoimmune reaction does occur in MG, it may perhaps be associated with a change in the proportions of T-cell subsets that lead to hyperfunction of the immune system. Many investigators have reported changes of T-cell subpopulations in patients with various diseases such as systemic lupus erythematosus (Alarcon-Segovia and Ruiz-Arguelles 1978), multiple sclerosis (Santoli et al. 1978), allergic disorders (Ong et al. 1979; Hsieh 1980) and myasthenia gravis (Shore et al. 1979). In this report the distributions of T-cell subpopulations were studied in MG patients and normal subjects to examine the possibility of abnormal immune regulation in MG. The changes of T-cell subsets after autologus serum incubation were also studied in order to test the possible role of serum factors in the pathogenesis of MG. MATERIALSAND METHODS
Patients and control subjects Thirty-eight MG patients (16 males and 22 females, aged 4-54 years) and 23 normal subjects (10 males and 13 females, aged 18-35 years) were included in this study. The patients had been diagnosed and treated at the Department of Neurology of National Taiwan University Hospital. All patients were receiving anticholinesterase drugs, and 23 patients were taking 60-120 mg of prednisolone on alternate days. Thymectomy had been performed in 7 patients; thymic hyperplasia was proved in 2 cases and thymoma in 1 case. The time interval between thymectomy and immunological studies ranged from 2 weeks to 76 months, with a mean of 27 months. Enumeration oJ"lymphocyte subpopulations Peripheral blood mononuclear cells were isolated by the method of Boyum (1968) and monocytes were removed by Petri dish adherence. T lymphocytes were purified by E rosetting and then centrifuged on Ficoll-Hypaque density gradient. The enumeration of B cells was made by EAC rosetting technique (Kerman et al. 1976), with slight modification (Hsieh 1980), and that of active T cells by the method of Wybran and Fudenberg (1973). Enumeration of T-cell subsets Preparation of IgM and IgG fractions of rabbit anti-Ox RBC antibodies and their Ox RBC-antibody complexes (EAM and EAG), and enumeration of T cells with receptors for IgM (T#) and IgG (Tv) were done according to the method of
55 Moretta et al. (1975), with slight modification (Hsieh 1980). Two hundred and fifty#1 of T lymphocyte suspension mixed with equal volume of serum was incubated in 37 °C water bath for 30 min, washed 3 times with HBSS, resuspended in 250/~1 of HBSS and then the T# and Ty cells were counted. Statistics The Student t-test was used for statistical analysis. RESULTS
The mean values of lymphocyte subpopulations, total lymphocyte count and T-cell subpopulations of MG patients and normal controls are shown in Table 1. There was no significant difference in the percentage of both T cells and B cells between patients and controls, but the mean of total lymphocyte count and of active T-cell percentage were marginally significantly lower in MG patients (0.05 < P < 0.1). The mean percentage of T/~ cells was significantly higher in MG patients than in controls (50.2 + 11.8~o vs. 38.5 + 15.3~, P < 0.01), whereas that of T7 cells in the former was lower than that in the latter (20.4 + 7.1~o vs. 27.6 + 6.2~, P < 0.001). The mean percentages of Tp cells and TV cells before and after autologus serum incubation were studied in 21 MG patients and 20 controls, and the results are shown in Table 2. Before incubation, the distribution of T-cell subsets of MG patients was quite different from that of controls in terms of increased T/~ cells (P < 0.01) and decreased TV cells ( P < 0.01); after incubation, the percentages of both T/~ and T? cells were decreased in MG patients, but only the decrease in T~,
TABLE 1 L Y M P H O C Y T E S U B P O P U L A T I O N S A N D T - C E L L S U B P O P U L A T I O N S IN M Y A S T H E N I A G R A V I S Total lymphocytes
B cells
T cells
Active T cells
T/~ cells
T? cells
( ~ of total lymphocytes)
(% o f total T cells)
673 + 333 (26.7 _+ 13.2)
1645 + 784 (64.5 _+ 9.5)
515 + 302 (19.7 -t- 4.8) b
(50.2 _+ 11.8)c
(20.4_+_7.1) d
799 + 183 (26.6 _+ 6.1)
1902 + 487 (63.3 _+ 9.5)
664 + 257 (22.1 _+ 4.8) b
(38.5 ___15.3) c
(27.6 +_ 6.2) d
Patients (n = 38) a.c. (/mm 3) %
2522 + 1036 *,a
N o r m a l s (n = 23) a.c. (/mm 3) %
3005 + 971 a
a.c. : absolute count. * Mean + SD. a 0.05
p<0.01;a
P<0.001.
56 TABLE 2 DISTRIBUTION OF T-CELL SUBPOPULATIONS BEFORE AND AFTER AUTOLOGUS SERUM INCUBATION IN MYASTHENIA GRAVIS T/~ (i',,)
T)' (I~o)
Before
After
Before
After
Patients (n = 21)
50,6 _+ 11.8*'~'
44.6 + 11.8
20.8 + 6.9 b'c
16.7 + 5.7~
Normals(n = 20)
38.0 _+ 14.5a
34.6 + 4.7
27.2 + 5.7~
26.5 _+4.2
* M e a n + SD. ~' P<0.01; b P<0.01; c P<0.05.
TABLE 3 TOTAL LYMPHOCYTE COUNT, TOTAL T-CELL COUNT AND T-CELL SUBPOPULATIONS lN DIFFERENTLY TREATED GROUPS OF MYASTHENIC PATIENTS Treated groups
Totallymphocyte count( /ram 3)
Total T-cell Tp (i~,j) count ( /ram 3)
T7 C~,,)
With steroids (n = 23) With anticholinesterase only (n = 15) Thymectomy (n = 7)
2725 _+ 1223" 2288_+ 663 2283 +_ 694
1771 _+923 1485 _+520 1475 _+506
19.6 _+7.2 21.8 _+7.0 24.8 +_5.8
47.6 +_ 10.6 54.6 +_ 12.6 5(/.3 _+ 13.4
* Mean +_ SD.
cells r e a c h e d a statistically s i g n i f i c a n t level ( P < 0.05). I n c o n t r a s t w i t h this, n o such c h a n g e was f o u n d in c o n t r o l s . T h e p e r c e n t a g e s o f T # cells a n d T~, cells in s t e r o i d - t r e a t e d , n o n - s t e r o i d t r e a t e d a n d t h y m e c t o m y g r o u p s are s h o w n in T a b l e 3. T h e r e was n o s i g n i f i c a n t difference in T/a cells a n d T7 cells b e t w e e n these g r o u p s , a l t h o u g h the steroidt r e a t e d g r o u p s h o w e d a t e n d e n c y to h a v e a s m a l l e r n u m b e r o f b o t h T/a a n d TV cells. DISCUSSION 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 u b p o p u l a t i o n s in M G p a t i e n t s h a v e b e e n r e p o r t e d b y several i n v e s t i g a t o r s ( A d b o u et al. 1974; K o z i n e r et al. 1976; C h e e et al. 1978; I t o y a m a et al. 1979), b u t the results a r e c o n f l i c t i n g . W e f o u n d t h a t the t o t a l c o u n t o f l y m p h o c y t e s a n d the p e r c e n t a g e o f active T cells were s m a l l e r in M G p a t i e n t s t h a n in n o r m a l c o n t r o l s ( m a r g i n a l l y s i g n i f i c a n t difference), w h e r e a s the c o u n t s o f T cells a n d B cells did n o t s h o w s i g n i f i c a n t difference b e t w e e n the two g r o u p s . A s active T cells h a v e b e e n c o n s i d e r e d to reflect m o r e closely the s t a t u s o f
57 cellular immunity, our observation of lower active T-cell count in patients may support the views of decreased mitogen response of T lymphocytes in MG patients studied by some investigators (Adner et al. 1966; Kawanami et al. 1979). The finding that there was no significant difference in the number of both T cells and active T cells between steroid-treated, non-steroid-treated and thymectomy groups was similar to that reported by Itoyama et al. (1979). T cells can be divided into two distinct subpopulations, T# cells and T~ cells. The T# cells, which comprise a large proportion of T cells up to 60~, express receptors for rabbit IgM and act as helpers for the proliferation and differentiation of B cells to plasma cells (Webb and Cooper 1973; Moretta et al. 1975, 1977; McConnell and Hurd 1976); whereas the T~ cells, which comprise a smaller proportion of T cells up to 15~o, express receptors for rabbit IgG and act as suppressors by interacting with IgG immune complexes (Greaves et al. 1974; Moretta et al. 1976a,b, 1977). Due to lack of a standardized method for performing T/~ and T7 rosette tests, the mean percentage of T/~ cells has been reported to range from 30.4 to 80~o and that of T), cells from 5 to 26~o (Moretta et al. 1975; Gupta and Good 1977, 1978, 1979). The mean values obtained in this study for T/~ cells (38.5~) and for T~ cells (27~) in healthy controls were within these ranges. Imbalances in the regulatory T-cell functions have been found in a variety of human diseases; increased suppressor T-cell activity as a cause of common variable immunodeficiency and malignancy, decreased suppressor T-cell function in the pathogenesis of autoimmune diseases (Waldman et al. 1978). Decreased T~ cells in patients with lupus erythematosus was reported by Alarcon-Segovia et al. (1978), who stated that the decrease was parallel to the disease activity. Shore et al. (1979) found hypofunction of suppressor T cells in MG patients by using plaque-forming cell assay. The decreased mitogen responsiveness of lymphocytes in MG patients was partly explained by the change of subpopulation of T lymphocytes (Kawanami et al. 1979). Galli et al. (1980) reported a case of MG associated with a monoclonal gammopathy which was considered to be a consequence of dysfunction of suppressor T lymphocytes. These lines of evidence are strongly suggestive of the possibility of impairment of suppressor T-cell function in MG patients. Moreover, there is some other evidence indicating the possibility of enhanced helper T-cell function in MG, such as increased autoantibody titers (Beutner et al. 1962; Peers et al. 1977) and increased EA(H)C-rosette-forming cells (Itoyama et al. 1979). Thymosin ~tl, which may perpetuate myasthenia gravis by facilitating overproduction of helper T lymphocytes, was also found in the sera of MG patients (Dalakas et al. 1980). Our findings of increased T/~ cells and decreased T7 cells further support the hypothesis of altered regulatory T-cell functions in the immunopathogenesis of myasthenia gravis. After incubation with autologus serum, the percentage of T~ cells was decreased in our MG patients, but no such a change was found in the normal controls. Shore et al. (1979) demonstrated a certain serum factor inhibiting Erosette formation in the IgG fraction of sera from MG patients. Fauci et al. (1978) suggested that decreased T7 cells in lupus patients might be due to the presence of
58 serum factors such as immune complexes which block the Fc receptors on T lymphocytes or anti-T-lymphocyte antibodies. Thus, in addition to the imbalance in T-cell regulatory functions, i.e. loss of suppressor and increase of helper T cells, the serum factors also appear to play a certain role in the immunopathogenesis of myasthenia gravis. ACKNOWLEDGEMENT
The authors are indebted to Dr. Rong-Chi Chen for sending sera of myasthenic patients for this study. REFERENCES Adbou, N. I., R. P. Lisak, B. Zweiman, I. Abrahamsohn and A. S. Penn (1974) The thymus in myasthenia gravis - - Evidence for altered cell populations, N. Engl. J. Med., 291 : 1271-1275. Adner, M.M., C. Isec, R.S. Schwab, J.D. Sherman and W. Dameshek (1966) Immunological studies of thymectomized and non-thymectomized patients with myasthenia gravis, Ann. N.Y. Acad. Sci., 135 : 536-548. Alarcon-Segovia, D. and A. Ruiz-Arguelles (1978) Decreased circulating thymus-derived cells with receptors for the Fc portion of immunoglobulin G in systemic lupus erythematosus, J. clin. Invest.. 62: 1390-1394. Almon, R. R., C.G. Andrew and S. H. Appel (1974) Serum globulin in myasthenia gravis inhibition of alpha-bungarotoxin binding to acetylcholine receptor, Science, 186: 55-57. Beutner, E. H., E. Witebsky, D. Ricken and R. H. Adler (1962) Studies on autoantibodies in myasthenia gravis, J. Amer. reed. Ass., 182: 46-48. Boyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood, Scand. J. clin. Lab. Invest. 21, Suppl. 97: 77-89. Chee, C.Y., R.C. Chen, T.P. Hung, T.S. Chen, G.H. Chen and J.S. Deng (1978) Immunological studies on Chinese myasthenia gravis, J. Formosan med. Ass., 77: 621-628. Chiu, H.C., R.C. Chen, T.P. Hung and J.S. Deng (1980) Leukocyte migration response to purified muscle antigen in myasthenia gravis patients, J. Formosan reed. Ass., 79: 977-985. Dalakas, M. C., W. K. Engel, J. E. McClure and A. L. Goldstein (1980) Thymosin ~t~ in myasthenia gravis, N. Engl. J. Med., 302: 1092-1093. Fauci, A. S., A.D. Steinberg, B.F. Haynes and G. Whalen (1978) Immunoregulatory aberrations in systemic lupus erythematosus, J. lmmunol., 121 : 1473-1479. Galli, M., G. Landi, D.L. Restelli and G. Scarlato (1980) Myasthenia gravis with a monoclonal gammopathy, J. neurol. Sci., 45: 103-108. Greaves, M., J. Janossy and M. Doenhoff (1974) Selective triggering of human T and B lymphocytes in vitro by polyclonal mitogens, J. exp. Med., 140: 1-18. Gupta, S. and R. A. Good (1977) Subpopulations of human T lymphocytes, Part 1 (Studies in immunodeficient patients), Clin. exp. Immunol., 30: 222-228. Gupta, S. and R. A. Good (1978) Subpopulations of human T lymphocytes, Part 5 (T lymphocytes with receptors for immunoglobulin M or G in patients with primary immunodeficiency disorders), Clin. Immunol. Immunopath., 11 : 292-302. Gupta, S. and R.A. Good (1979) Subpopulations of human lymphocytes, Part 10 (Alterations in T.B. third population cells and T cells with receptors for immunoglobulin M (T#) or G (T~) in aging human, J. Immunol., 122: 1214-1219. Hsieh, K.H. (1980) Different responses of T-cell subpopulations to allergen challenge in asthmatic children and normals, Ann. Allergy, 44: 177-180. ltoyama, Y., S. Kawanami, T. Goto and Y. Kuroiwa (1979) T and B lymphocytes in myasthenia gravis, Clin. exp. lmmunol., 37: 452-456. Kawanami, S., A. Kanaide, Y. Itoyama and Y. Kuroiwa (1979) Lymphocyte function in myasthenia gravis, J. Neurol. Neurosurg. Psychiat., 42: 734-740. Kerman, R., R. Smith, E. Ezdinili and S. Stefani (1976) Unification and technical aspects of total T, active T and B lymphocyte rosette assay, Immunol. Commun., 5 : 685-694.
59 Kott, E., G. Genkins and A. H. Rule (1973) Leukocyte response to muscle antigens in myasthenia gravis, Neurology (Minneap.), 23 : 374-380. Koziner, B., K.J. Block and U.P. Perlo (1976) Distribution of peripheral blood latex-ingesting cells, T-cells and B-cells in patients with myasthenia gravis, Ann. N. Y. Acad. Sci., 274:411-420. Lindstrom, J.A., M.E. Seybold, V.A. Lennon, S. Whittingham and D.D. Duane (1976) Antibody to acetylcholine receptor in myasthenia gravis - - Prevalence, clinical correlates and diagnostic value, Neurology ( Minneap. ) , 26: 1054-1059. McConnell, I. and C. M. Hurd (1976) Lymphocyte receptors, Part 2 (Receptors for rabbit IgM on human T lymphocytes), Immunology, 30: 835-839. Moretta, L., M. Ferrarini, M. L. Durante and M. C. Mingari (1975) Expression of a receptor for IgM by human T cells in vitro, Europ. J. Immunol., 5 : 565-569. Moretta, L., M. Ferrarini, M. C. Mingari, A. Moretta and S. R. Webb (1976a) Subpopulations of human T cells identified by receptors for immunoglobulins and mitogen responsiveness, J. Immunol., 117: 2171-2174. Moretta, L., S. R. Webb, C. E. Grossi, P. M. Lydyard and M. D. Cooper (1976b) Functional analysis of two subpopulations of human T cells and their distribution in immunodeficient patients, Clin. Res., 24:448 (Abstract). Moretta, L., S.R. Webb, C. E. Grossi, P. M. Lydyard and M. D. Cooper (1977) Functional analysis of two human T-cell subpopulations - - Help and suppression of B-cell responses by T cells bearing receptors for IgM or IgG, .L exp. Med., 146: 184-200. Ong, K.S., M.H. Grieco and Z. Goel (1979) Responses in vitro to antigen E by T lymphocytes of subjects sensitive and nonsensitive to ragweed, J. Allerg. clin. Immunol., 63 : 149 (Abstract). Peers, J., B.L. McDonald and R.L. Dawkins (1977) The reactivity of the antistriational antibodies associated with thymoma and myasthenia gravis, Clin. exp. Immunol., 27: 66-73. Santoli, D., L. Moretta, R. Lisak, D. Gilden and H. Koproski (1978) Imbalances in T cell subpopulations in multiple sclerosis patients, J. lmmunol., 120: 1369-1371. Shirai, T., M. Miyata, A. Nakase and T. Itoh (1976) Lymphocyte subpopulation in neoplastic and nonneoplastic thymus and in blood of patients with myasthenia, Clin. exp. Immunol., 26:118-123. Shore, A., S. Limatibul, H. M. Dosch and E. W. Gelfand (1979) Identification of two serum components regulating the expression ofT-lymphocyte function in children myasthenia gravis, N. Engl. J. Med., 301: 625--629. Simpson, J. A. (1966) Myasthenia gravis as an autoimmune disease - - Clinical aspects, Ann. N.Y. Acad. Sci., 135: 506-516. Waldman, T.A., R.M. Blase, S. Broder and R.S. Krakauer (1978) Disorders of suppressor immunoregulatory cells in the pathogenesis of immunodeficiency and autoimmunity, Ann. int. Med., 88 : 226-238. Webb, S.R. and M.D. Cooper (1973) T cells can bind antigen via cytophilic IgM antibody made by B cells, J. Immunol., 111 : 275-277. Wybran, J. and H. H. Fundenberg (1973) Thymus derived rosette-forming cells in various human disease states, J. clin. Invest., 52: 1026-1032.
53
IMBALANCES IN T-CELL SUBPOPULATIONS IN MYASTHENIA GRAVIS
HOU-CHANG CHIU l, KUE-HSIUNG HSIEH 2 and TSU-PEI HUNG ~
IDepartment of Neurology and 2pediatrics, National Taiwan University Hospital, Taipei, Taiwan 100 (Republic of China) (Received 10 December, 1980) (Revised, received 12 March, 1981) (Accepted 19 March, 1981)
SUMMARY
Distributions of peripheral blood lymphocyte subpopulations and T-cell subsets were studied in 38 patients with myasthenia gravis (MG) and 23 healthy controls. T cells were detected by rosette formation with sheep red blood cells and B cells with erythrocyte-antibody-complement (EAC) complexes. T# cells were identified by rosette formation of T cells with Ox RBC-IgM complexes, and Ty cells with Ox RBC-IgG complexes. The means of total lymphocyte count and active T cell percentage were marginally significantly lower in MG patients than in normal controls (0.05
INTRODUCTION
Myasthenia gravis (MG) has been considered as an autoimmune disease on the basis of clinical manifestations (Simpson 1966) and laboratory findings (Kott This study was supported by a grant from the National Council of Science, Republic of China ; grant No. NSC-70B-0412-02(04). Address for correspondence: Dr. Hou-Chang Chiu, Department of Neurology, National Taiwan University Hospital, 1, Chang-Te Street, Taipei, Taiwan 100, Republic of China. 0022-510X/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
54 et al. 1973; Almon et al. 1974; Lindstrom et al. 1976). Abnormal lymphocyte functions in MG have been reported by several investigators (Kawanami et al. 1979; Chiu et al. 1980). Although no significant difference in peripheral blood lymphocyte subpopulations between MG patients and control subjects was found (Shirai et al. 1976; Chee et al. 1978), the normal counts could not exclude the possibility of quantitative and/or qualitative abnormalities of T cell subpopulations in MG patients. If an autoimmune reaction does occur in MG, it may perhaps be associated with a change in the proportions of T-cell subsets that lead to hyperfunction of the immune system. Many investigators have reported changes of T-cell subpopulations in patients with various diseases such as systemic lupus erythematosus (Alarcon-Segovia and Ruiz-Arguelles 1978), multiple sclerosis (Santoli et al. 1978), allergic disorders (Ong et al. 1979; Hsieh 1980) and myasthenia gravis (Shore et al. 1979). In this report the distributions of T-cell subpopulations were studied in MG patients and normal subjects to examine the possibility of abnormal immune regulation in MG. The changes of T-cell subsets after autologus serum incubation were also studied in order to test the possible role of serum factors in the pathogenesis of MG. MATERIALSAND METHODS
Patients and control subjects Thirty-eight MG patients (16 males and 22 females, aged 4-54 years) and 23 normal subjects (10 males and 13 females, aged 18-35 years) were included in this study. The patients had been diagnosed and treated at the Department of Neurology of National Taiwan University Hospital. All patients were receiving anticholinesterase drugs, and 23 patients were taking 60-120 mg of prednisolone on alternate days. Thymectomy had been performed in 7 patients; thymic hyperplasia was proved in 2 cases and thymoma in 1 case. The time interval between thymectomy and immunological studies ranged from 2 weeks to 76 months, with a mean of 27 months. Enumeration oJ"lymphocyte subpopulations Peripheral blood mononuclear cells were isolated by the method of Boyum (1968) and monocytes were removed by Petri dish adherence. T lymphocytes were purified by E rosetting and then centrifuged on Ficoll-Hypaque density gradient. The enumeration of B cells was made by EAC rosetting technique (Kerman et al. 1976), with slight modification (Hsieh 1980), and that of active T cells by the method of Wybran and Fudenberg (1973). Enumeration of T-cell subsets Preparation of IgM and IgG fractions of rabbit anti-Ox RBC antibodies and their Ox RBC-antibody complexes (EAM and EAG), and enumeration of T cells with receptors for IgM (T#) and IgG (Tv) were done according to the method of
55 Moretta et al. (1975), with slight modification (Hsieh 1980). Two hundred and fifty#1 of T lymphocyte suspension mixed with equal volume of serum was incubated in 37 °C water bath for 30 min, washed 3 times with HBSS, resuspended in 250/~1 of HBSS and then the T# and Ty cells were counted. Statistics The Student t-test was used for statistical analysis. RESULTS
The mean values of lymphocyte subpopulations, total lymphocyte count and T-cell subpopulations of MG patients and normal controls are shown in Table 1. There was no significant difference in the percentage of both T cells and B cells between patients and controls, but the mean of total lymphocyte count and of active T-cell percentage were marginally significantly lower in MG patients (0.05 < P < 0.1). The mean percentage of T/~ cells was significantly higher in MG patients than in controls (50.2 + 11.8~o vs. 38.5 + 15.3~, P < 0.01), whereas that of T7 cells in the former was lower than that in the latter (20.4 + 7.1~o vs. 27.6 + 6.2~, P < 0.001). The mean percentages of Tp cells and TV cells before and after autologus serum incubation were studied in 21 MG patients and 20 controls, and the results are shown in Table 2. Before incubation, the distribution of T-cell subsets of MG patients was quite different from that of controls in terms of increased T/~ cells (P < 0.01) and decreased TV cells ( P < 0.01); after incubation, the percentages of both T/~ and T? cells were decreased in MG patients, but only the decrease in T~,
TABLE 1 L Y M P H O C Y T E S U B P O P U L A T I O N S A N D T - C E L L S U B P O P U L A T I O N S IN M Y A S T H E N I A G R A V I S Total lymphocytes
B cells
T cells
Active T cells
T/~ cells
T? cells
( ~ of total lymphocytes)
(% o f total T cells)
673 + 333 (26.7 _+ 13.2)
1645 + 784 (64.5 _+ 9.5)
515 + 302 (19.7 -t- 4.8) b
(50.2 _+ 11.8)c
(20.4_+_7.1) d
799 + 183 (26.6 _+ 6.1)
1902 + 487 (63.3 _+ 9.5)
664 + 257 (22.1 _+ 4.8) b
(38.5 ___15.3) c
(27.6 +_ 6.2) d
Patients (n = 38) a.c. (/mm 3) %
2522 + 1036 *,a
N o r m a l s (n = 23) a.c. (/mm 3) %
3005 + 971 a
a.c. : absolute count. * Mean + SD. a 0.05
p<0.01;a
P<0.001.
56 TABLE 2 DISTRIBUTION OF T-CELL SUBPOPULATIONS BEFORE AND AFTER AUTOLOGUS SERUM INCUBATION IN MYASTHENIA GRAVIS T/~ (i',,)
T)' (I~o)
Before
After
Before
After
Patients (n = 21)
50,6 _+ 11.8*'~'
44.6 + 11.8
20.8 + 6.9 b'c
16.7 + 5.7~
Normals(n = 20)
38.0 _+ 14.5a
34.6 + 4.7
27.2 + 5.7~
26.5 _+4.2
* M e a n + SD. ~' P<0.01; b P<0.01; c P<0.05.
TABLE 3 TOTAL LYMPHOCYTE COUNT, TOTAL T-CELL COUNT AND T-CELL SUBPOPULATIONS lN DIFFERENTLY TREATED GROUPS OF MYASTHENIC PATIENTS Treated groups
Totallymphocyte count( /ram 3)
Total T-cell Tp (i~,j) count ( /ram 3)
T7 C~,,)
With steroids (n = 23) With anticholinesterase only (n = 15) Thymectomy (n = 7)
2725 _+ 1223" 2288_+ 663 2283 +_ 694
1771 _+923 1485 _+520 1475 _+506
19.6 _+7.2 21.8 _+7.0 24.8 +_5.8
47.6 +_ 10.6 54.6 +_ 12.6 5(/.3 _+ 13.4
* Mean +_ SD.
cells r e a c h e d a statistically s i g n i f i c a n t level ( P < 0.05). I n c o n t r a s t w i t h this, n o such c h a n g e was f o u n d in c o n t r o l s . T h e p e r c e n t a g e s o f T # cells a n d T~, cells in s t e r o i d - t r e a t e d , n o n - s t e r o i d t r e a t e d a n d t h y m e c t o m y g r o u p s are s h o w n in T a b l e 3. T h e r e was n o s i g n i f i c a n t difference in T/a cells a n d T7 cells b e t w e e n these g r o u p s , a l t h o u g h the steroidt r e a t e d g r o u p s h o w e d a t e n d e n c y to h a v e a s m a l l e r n u m b e r o f b o t h T/a a n d TV cells. DISCUSSION 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 u b p o p u l a t i o n s in M G p a t i e n t s h a v e b e e n r e p o r t e d b y several i n v e s t i g a t o r s ( A d b o u et al. 1974; K o z i n e r et al. 1976; C h e e et al. 1978; I t o y a m a et al. 1979), b u t the results a r e c o n f l i c t i n g . W e f o u n d t h a t the t o t a l c o u n t o f l y m p h o c y t e s a n d the p e r c e n t a g e o f active T cells were s m a l l e r in M G p a t i e n t s t h a n in n o r m a l c o n t r o l s ( m a r g i n a l l y s i g n i f i c a n t difference), w h e r e a s the c o u n t s o f T cells a n d B cells did n o t s h o w s i g n i f i c a n t difference b e t w e e n the two g r o u p s . A s active T cells h a v e b e e n c o n s i d e r e d to reflect m o r e closely the s t a t u s o f
57 cellular immunity, our observation of lower active T-cell count in patients may support the views of decreased mitogen response of T lymphocytes in MG patients studied by some investigators (Adner et al. 1966; Kawanami et al. 1979). The finding that there was no significant difference in the number of both T cells and active T cells between steroid-treated, non-steroid-treated and thymectomy groups was similar to that reported by Itoyama et al. (1979). T cells can be divided into two distinct subpopulations, T# cells and T~ cells. The T# cells, which comprise a large proportion of T cells up to 60~, express receptors for rabbit IgM and act as helpers for the proliferation and differentiation of B cells to plasma cells (Webb and Cooper 1973; Moretta et al. 1975, 1977; McConnell and Hurd 1976); whereas the T~ cells, which comprise a smaller proportion of T cells up to 15~o, express receptors for rabbit IgG and act as suppressors by interacting with IgG immune complexes (Greaves et al. 1974; Moretta et al. 1976a,b, 1977). Due to lack of a standardized method for performing T/~ and T7 rosette tests, the mean percentage of T/~ cells has been reported to range from 30.4 to 80~o and that of T), cells from 5 to 26~o (Moretta et al. 1975; Gupta and Good 1977, 1978, 1979). The mean values obtained in this study for T/~ cells (38.5~) and for T~ cells (27~) in healthy controls were within these ranges. Imbalances in the regulatory T-cell functions have been found in a variety of human diseases; increased suppressor T-cell activity as a cause of common variable immunodeficiency and malignancy, decreased suppressor T-cell function in the pathogenesis of autoimmune diseases (Waldman et al. 1978). Decreased T~ cells in patients with lupus erythematosus was reported by Alarcon-Segovia et al. (1978), who stated that the decrease was parallel to the disease activity. Shore et al. (1979) found hypofunction of suppressor T cells in MG patients by using plaque-forming cell assay. The decreased mitogen responsiveness of lymphocytes in MG patients was partly explained by the change of subpopulation of T lymphocytes (Kawanami et al. 1979). Galli et al. (1980) reported a case of MG associated with a monoclonal gammopathy which was considered to be a consequence of dysfunction of suppressor T lymphocytes. These lines of evidence are strongly suggestive of the possibility of impairment of suppressor T-cell function in MG patients. Moreover, there is some other evidence indicating the possibility of enhanced helper T-cell function in MG, such as increased autoantibody titers (Beutner et al. 1962; Peers et al. 1977) and increased EA(H)C-rosette-forming cells (Itoyama et al. 1979). Thymosin ~tl, which may perpetuate myasthenia gravis by facilitating overproduction of helper T lymphocytes, was also found in the sera of MG patients (Dalakas et al. 1980). Our findings of increased T/~ cells and decreased T7 cells further support the hypothesis of altered regulatory T-cell functions in the immunopathogenesis of myasthenia gravis. After incubation with autologus serum, the percentage of T~ cells was decreased in our MG patients, but no such a change was found in the normal controls. Shore et al. (1979) demonstrated a certain serum factor inhibiting Erosette formation in the IgG fraction of sera from MG patients. Fauci et al. (1978) suggested that decreased T7 cells in lupus patients might be due to the presence of
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