Abnormalities of T-cell subpopulations in the blood and thymus of patients with myasthenia gravis

Journal of the Neurological Sciences, 1979, 44: 69-76

69

:(~ Elsevier/North-Holland Biomedical Press

A B N O R M A L I T I E S OF T-CELL S U B P O P U L A T I O N S IN T H E BLOOD A N D T H Y M U S OF P A T | E N T S W I T H M Y A S T H E N I A G R A V I S

ROBERT P. LISAK, ROSE SMILEY, DONALD H. SCHOTLAND, WILLIAM J. BANK Jr. and DANIELA SANTOLI Hem3' M. Watts, Jr. Muscular Dystrophy Association Neuromuscular Research Center, Department 0] Neurology of the University of Pennsylvania and the Wistar Institute- University of'Pennsylvania Multiple Sclerosis Research Center, Philadelphia, PA 19104 (U.S.A.)

(Received 8 May, 1979) (Accepted 11 June, 1979)

SUMMARY Subpopulations of T cells were quantitated in the blood and thymus of patients with myasthenia gravis. As compared to healthy donors, an increase in T lymphocytes bearing receptors for IgG (TG) was found in the blood of 10 of 28 patients. T~ and TM (T cells bearing receptors for IgM) cells were found in thymuses without thymoma.

INTRODUCTION Myasthenia gravis is an immunologically mediated disease of unknown etiology (Simpson 1960; Lisak 1975; Simpson 1977, 1978; Drachman 1978). The co-existence of other putative autoimmune diseases (Simpson 1960; Downes et al. 1966) and autoantibodies (Adner et al. 1964; Downes et al. 1966; Penn et al. 1971) in the serum of patients with myastenia gravis suggests the possibility that a loss of normal immunoregulatory control mechanisms may be important in the development of this disease (Lisak 1975; Simpson et al. 1976). In addition, the presence of histologic changes in the thymus including thymoma (Castleman 1966) suggests a more widespread dysfunction in the immunologic system. If myasthenia is accompanied by a defect in the immune system, there might be a change in the subpopulations of immunocompetent cells in the blood and/or thymus.

This work was supported, in part, by NS-11036 from the National Institute of Neurologic and Communicative Diseases and Stroke, by the National Multiple Sclerosis Society, by United States Public Health Service Research Grants CA-20833 and CA-21069 from the National Cancer Institute and by the Muscular Dystrophy Association.

70 Recently, it has been shown that human T (thymic-dependent) lymphocytes can be separated on the basis of ability to bind either IgG or IgM immune complexes (Dickler et al. 1974; Ferrarini et al. 1975; Moretta et al. 1976, 1978). Up to 75~ of human T cells have Fc receptors for IgM (TM); a smaller proportion (5-20 ~) has Fc receptors for IgG (TG). The remainder of T cells lack detectable receptors for either immunoglobulin (T "null"). Lymphocytes in the human post-natal thymus consist almost entirely of T "null" cells (Moretta et al. 1978). MATERIALSAND METHODS

Patient population Peripheral blood. Forty-eight coded peripheral blood samples were obtained from 28 patients with myasthenia gravis. Normals consisted of two separate groups of healthy volunteers, also examined and coded. Peripheral blood lymphocytes (PBL) were obtained by Ficoll-Hypaque centrifugation and removal of adherent cells on plastic flasks (Santoli et al. 1978a). Clinical status. The clinical severity of patients with myasthenia was determined without knowledge of the results of laboratory findings and classified as follows: (a) remission : without symptoms of myasthenia and on no or minimal doses of medications; (b) mild: slight symptoms of myasthenia requiring either no or minimal doses of medication; (c) moderate: symptoms of myasthenia of a degree to interfere with normal activities despite optimal doses of current medications; (d) severe: very marked restriction of activities due to myasthenia; (e)crisis: severe impairment of bulbar and respiratory functions requiring ventilatory assistance. Thymus. Suspensions of thymic lymphocytes (TL) were made from tissue obtained at therapeutic thymectomy (Abdou et al. 1974; Lisak et al. 1976) from 6 patients with myasthenia gravis: 2 with thymic hyperplasia with germinal follicles, 2 with lymphoepithelial thymoma, and 2 with normal histology. Enumeration ofT and T-cell subsets. The methods used to quantitate T and T-cell subsets have been recently described in detail (Moretta et al. 1977; Santoli et al. 1978a). Briefly, T cells from PBL and TL were separated on the basis of their ability to form E rosettes with neuraminidase-treated sheep erythrocytes. T cell subsets were then separated from the purified T-cell fraction using a rosette technique with ox erythrocytes coated with highly purified IgG (EA-IgG) or IgM (EA-IgM) antibodies. Enumeration of EA-IgG and EA-IgM rosettes gave the percentage of TG and T~ cells, respectively. TM cells were always quantitated after overnight incubation of T lymphocytes (Moretta et al. 1976) whereas TG cells were counted immediately after purification of T cells in most cases. The average percentage of E, EA-IgG and EA-IgM rosettes was determined in triplicate samples after counting at least 600 viable cells. RESULTS

Peripheral blood lymphocytes (PBL) An increase in the percentage of TG cells in the blood of 10 of the 28 patients

71 TABLE 1 PERCENTAGE OF TOTAL T CELLS AND T-CELL SUBSETS 1N NORMAL SUBJECTS Source

Number

Ta

To a

T~ ~

Bloodl) Blood Thymusa

10 49 4

66.8 ± 1.1 69.0 ± 1.0 98.0 5- 1.0

12.4 ± 1.2 12.1 ± 1.0 1.0 ± 1.0

NT e 56.0 ± 2.0 3.0 ± 2.0

'~ Mean ~ SE. Total T cells are expressed as percentage of lymphocytes and Tc~ and T~t cells as percentage of T cells. b From Santoli et al. (1978a). e Not tested. ~ From Moretta et al. (1978). Thymic cell suspensions were obtained at thoracotomy from 4 adult subjects without known immunologic disease. with myasthenia was observed when compared to the two different groups ofnormals (~> 2 SD above the mean of normals: Tables 1-4). Of these 10 myasthenic patients, only one (patient 20) had undergone thymectomy more than two years prior to study (Tables 3 and 4). Twelve patients were studied sequentially (Table 3). Marked fluctuations in the levels of T6 cells were observed in some of these subjects, whereas no meaningful variation was seen in serially studied normals (Table 2). A considerable variability in TG cells was seen in myasthenic patients after thymectomy (Table 3). In one there was a transient rise followed by a subsequent fall to normal range (patient I). In another subject (patient 5) there was a decrease followed by a temporary increase back to an elevated level and then a subsequent decline into a normal range. In two subjects with clearly normal pre-operative levels, there was no change 1 week after thymectomy (patients 4 and 6). Patient 8, who underwent thymectomy for a thymoma without any short-term clinical benefit and who required the addition of prednisone, had an increase in TG cells. An additional patient (patient 10) showed a decrease from an elevated level to normal on two occasions while under treatment with prednisone. Two serially studied patients (3 and 9) did not undergo thymectomy during the period of study. One of them (3) improved clinically with anticholinesterase therapy and plasmapheresis without the use of prednisone or cytotoxic agents. He showed a decrease in the percentage of To cells, although not to normal levels. Three other patients (7, 11 and 12) had already undergone thymectomy before the study and although all three showed decreasing levels of TG cells with time, the initial postthymectomy level examined was not clearly elevated in any of them. No abnormalities were noted in the percentage of TM cells in the PBL of any patient with myasthenia gravis.

Thymic lymphocytes (TL) As compared to data reported by Moretta et al. (1978), a mild decrease in the percentage o f T cells was detectable in the thymus of the patients tested (Tables 1 and 5). No difference in the percentage of either T-cell subpopulation was seen between thymoma specimens (Table 5) and thymus obtained from control thoracotomy

72 TABLE 2 PERCENTAGE OF TOTAL T CELLS AND T-CELL SUBSETS IN THE BLOOD OF NORMAL SUBJECTS: SERIAL STUDIES Subject

Ta

T~ a

T~ a

Comment

A

58 54 61

10 13 13

60 58 71

1st study 28 days 15No. 1 7 days 15 No. 2

B

54 63 76 62 60 70

11 18 19 16 15 12

NT b 65 53 62 75 65

1st study 7 days 15No. 7 days 15No. 7 days ~ No. 21 days 15 No. 14 days 15No.

C

72 63 58

4 7 9

68 71 59

1st study 27 days 15No. 1 21 days 15 No. 2

D

53 63 55 65 53

10 11 5 4 12

70 NT 59 NT 64

1st study 3 mos ~ No. 1 14 days 15 No. 2 21/2 mos 15No. 3 27 days ~ No. 4

E

NT 55 60 NT 71

7 13 12 11 7

64 50 53 55 63

1st study 14 days ~ No. 1 7 days ~ No. 2 7 days ~ No. 3 1 mo 15 No. 4

F

68 72 64 71

6 4 7 5

NT 61 55 58

1st study 5 wks 15No. 1 14 days ~ No. 2 20 days 15 No. 3

G

66 55 72

8 4 5

51 NT 63

1st study 4 wks ~ No. 1 1 wk~No. 2

H

61 62 74

9 4 5

55 68 NT

1st study 6 wks 15No. 1 1 wk ~ No. 2

I

NT 76 58 75

6 10 9 9

57 65 62 68

1st study 2 wks 15No. 1 3 wks 15No. 2 2 wks 15 No. 3

a Mean ± SE. b Not tested.

1 2 3 4 5

73 TABLE 3 P E R C E N T A G E S O F T O T A L T C E L L S A N D T - C E L L S U B S E T S IN B L O O D O F P A T I E N T S WITH MYASTHENIA GRAVIS: SERIAL STUDIES Patient

1

Age (yr)

Sex

T

T~;

TM

Status a

R~ b

C o m m e n t ''

23

F

58 54 70 65

27 43 24 4

53 52 61 69

moderate moderate mild remission

AC AC AC AC

fi Tx 13 days 15 Tx (H) 23 days ~ Tx 4 m o s ff Tx

28

M

59 51

30 9

NT d 71

mild mild

AC AC

fiTx 1 wk p Tx (N)

74

M

52 69 63 76

43 35 27 25

50 58 51 NT

severe severe moderate remission

AC AC AC AC

30

F

58 62

14 9

50 51

moderate moderate

AC AC

fi Tx 1 wk ~ Tx (H)

25

F

63 60 73 62

19 12 6 25

64 70 63 NT

moderate moderate mild mild

AC AC AC AC

5. Tx I m o ~ Tx (H) 2 m o s ~ Tx 6 m o s 15 Tx

54

17

59

mild

AC

I st study ( N o Tx) 3 wks. later 3 m o s later 8 m o s later

8 m o s ~ Tx

6

28

F

68 61

9 8

64 61

moderate moderate

AC ; P AC; P

5. Tx I wk 15 Tx (H)

7

29

F

NT 64

18 6

58 NT

mild mild

AC AC

3 yrs ~ T x ( H ) 31/2 yrs ~ Tx

8

31

F

58 36

17 26

59 NT

severe severe

AC AC; P

5_ Tx 4 m o s ~ Tx (Th)

9

69

F

NT 62

17 13

61 67

crisis moderate

AC A C ; P; Az

Ist study 7 m o s later

10

29

M

70 70 52

25 4 11

42 58 NT

moderate moderate moderate

AC AC; P AC; P

1st s t u d y 5 m o s later 3 days later - day o f T x (N)

11

48

F

45 59

21 8

60 64

severe remission

AC; P AC; P

5 m o s 15 T X (Th) 4 m o s later

12

59

F

66 60

18 9

70 NT

moderate mild

AC AC

11/2 yrs ~ Tx (Th) 2 yrs ~ Tx

Clinical status at time of study. b Medications: A C -- anticholinesterase; P p r e d n i s o n e ; Az azathioprine. " 5. Tx before t h y m e c t o m y ; 15 Tx - after t h y m e c t o m y ; N normal thymus; H with germinal follicles; T h thymoma. b N T -- not tested.

"hyperplasia"

74 TABLE 4 P E R C E N T A G E O F T O T A L T CELLS A N D T-CELL SUBSETS IN B L O O D OF P A T I E N T S W I T H M Y A S T H E N I A GRAVIS Patient

Age (yr)

Sex

T

TG

TM

Status

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

65 46 29 63 46 67 65 25 60 28 46 43 54 32 18 63

F F F M F M M F M F F M F M F F

NT 64 58 75 72 48 60 45 51 51 56 62 63 58 57 50

14 14 21 20 35 15 6 22 8 6 3 9 16 17 7 8

58 moderate 60 crisis 61 mild 62 moderate NT moderate 55 mild 69 mild 51 moderate N T e mild 68 moderate 61 mild 59 remission 65 mild 51 mild 58 moderate 62 moderate

Rx a

AC AC AC AC AC AC; AC AC AC AC; AC AC; AC AC AC; AC;

Commentb

no Tx 3 yrs ~ Tx (N) 1 year ~ Tx (H) h Tx (N) 6 mos. ~ Tx (Th) P ~ Tx (Th) 1 year ~ Tx (N) 3 yrs. ~ Tx (H) no Tx P; Cy 3 yrs 1) Tx (Th) no Tx P no Tx no Tx 2 yrs ~ Tx (Th) P 13 days ~ Tx (N) P no Tx

a Medications: Cy = cyclophosphamide and see Table 3. b See Table 3. e N T = not tested.

subjects (Table 1). In contrast, high levels of TG and TM cells were observed in two (patients 1 and 2) of 4 TL suspensions obtained from non-thymoma specimens. In the other two such TL suspensions a slight increase of TM (patient 6) or TG (patient 10) was observed. Patient 10 had been receiving prednisone for several months prior to thymectomy. DISCUSSION

Although these observations are preliminary, the results suggest that an increase TABLE 5 P E R C E N T A G E S O F T CELLS A N D T-CELL SUBSETS IN T H Y M U S OF PATIENTS W I T H M Y A S T H E N I A GRAVIS Patient

Age (yr)

Sex

T

TG

TM

Histology a

Status a

Rx a

1 2 6 8 10 29

23 28 28 31 29 37

F M F F M F

92 91 93 NT 80 NT

19 10 2 0.5 5 1

30 65 9 1 0.5 1

H N H Th N Th

moderate mild moderate severe moderate moderate

AC AC AC AC AC; P AC

a See Table 3.

75 in the percentage of circulating T~ cells occurs in some patients with myasthenia gravis during the course of their disease. Birnbaum and Tsairis (1976) reported a decrease in in vitro suppressor cell activity in myasthenic patients who had undergone thymectomy. More recently, a decrease in in vitro suppressor activity in myasthenia has been reported by other investigators but relation to therapy and clinical status was not detailed (Mischak et al. 1978). These observations may relate to our findings since T6 cells include lymphocytes with in vitro suppressor activity (Moretta et al. 1977). It should be noted, however, that T6 lymphocytes also have cytotoxic activity (Santoli et al. 1978b). It is not known as yet whether the same T6 cell is responsible for both suppressor and cytotoxic activity or whether these functions are properties of distinct T , cells. No conclusions can be drawn from the present study about the possible effects of prednisone on the distribution of circulating T cell subsets in myasthenic patients. Ten patients were studied while receiving prednisone (Tables 3 and 4); however, only 5 were among the serial patients and only one patient (patient 10) was studied (a) both before and during therapy, and (b) without concomitant thymectomy or cytotoxic immunosuppressive agents. The observation of an increase in the percentage of both T6 and TM cells in the thymuses of some patients with myasthenia without thymoma can be considered further evidence for abnormalities of thymic lymphocytes. Among such previously described abnormalities are: (a) an increase in B tymphocytes (Abdou et al. 1974; Lisak et al. 1976; Shirai et al. 1976; Cook et al. 1977; Opelz et al. 1978; Lisak et al. 1978); (b) a mild decrease in the percentage o f T cells (Abdou et al. 1974; Lisak et al. 1976; Trotter et al. 1977: Lisak et al. 1978); (c) increased in vitro proliferative response induced by pokeweed mitogen (Abdou et al. 1974); (d) increased in vitro immunoglobulin production (Smiley et al. 1969); (e) in vitro synthesis of antibody to acetylcholine receptor (Vincent et al. 1978); and (g) an in vitro autologous mixed leukocyte reaction betweern PBL and TL (Abdou et al. 1974; Lisak et al. 1976: Trotter et al. 1977: Opelz et al. 1978). The possible role of the changes in T-cell subsets in the PBL and TL of patients with myasthenia gravis is not clear. Although the increase in levels of T6 cells in the examined patients did not seem to correlate with any particular stage of disease, further studies in a larger number of serial patients might help to establish a correlation between clinical status and levels of circulating T6 cells. Such studies might also shed light on the effects, if any, of thymectomy, corticosteroids (Fauci et al. 1978) and cytotoxic immunosuppressive agents. In addition, in vitro functional studies are currently being undertaken in our laboratory to examine both cytotoxic and suppressor activities of TG cells of myasthenia gravis patients as well as their interaction with acetylcholine receptor. Note: Since this manuscript was submitted an increase in blood T6 cells was reported in some patients with myasthenia gravis (Piantelli et al. 1979).

76 REFERENCES Abdou, N. I., R. P. Lisak, B. Zweiman, 1. 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., J. D. Sherman, C. Ise, R. S. Schwab and W. Dameshek (1964) An immunologic survey of forty-eight patients with myasthenia gravis, N. Er.gl. J. Med., 27l : 1327-1333. Birnbaum, G. and P. Tsairis (1976) Suppressor lymphocytes in myasthenia gravis and effect of adult thymectomy, Ann. N. Y. Acad. Sci., 274: 527-535. Castleman, B. (1966) The pathology of the thymus gland in myasthenia gravis, Ann. N. Y. ,4cad. Sci., 1 3 5 : 496-505. Cook, J. D., J. L. Trotter, W. K. Engel and C. L. Mclntosh (1977) Altered immunologic cell populations in thymuses from myasthenia gravis patients, Neurology (Minneap.), 11 : 365. Dickler, H. B., N. F. Adkinson Jr. and W. D. Terry (1974) Evidence for individual human l~eripheral blood lymphocytes bearing both B and T cell markers, Nature (Lond.), 247: 213-215. Downes, J. A., B. M. Greenwood and S. H. Wray (1966) Auto-immune aspects of myasthenia gravis, Quart. J. Med., 35: 85-105. Drachman, D. B. (1978) Myasthenia gravis, N. Engl. J. Med., 298: 136-142. Fauci, A. S., A. D. Steinberg, B. F. Haynes and G. Whalen (1978) Immunoregulatory aberrations in systemic lupus erythematosus, J. lmmunol., 121 : 1473-1479. Ferrarini, M., L. Moretta, R. Abrile and M. L. Durante (1975) Receptors for lgG molecules on human lymphocytes forming spontaneous rosettes with sheep red cells, Europ. J. lmmunoL, 5: 70-72. Lisak, R. P. (1975) Immunologic asl:eCtS of myasthenia gravis, Ann. elin. Lab. Sci., 5: 228-293. Lisak, R. P., B. Zweiman and S. M. Phillips (1978) Thymic and peripheral blood T and B cell levels in myasthenia gravis, Neurology (Minneap.), 28: 1298-1301, Lisak, R. P., N. I. Abdou, B. Zweiman, C. Zmijewski and A. S. Penn (1976) Aspects of lymphocyte function in myasthenia gravis, Ann. N. Y. Acad. Sci., 274: 402-411. Mischak, R. P., P. C. Dau, R. L. Gonzales and L. E. Spitler (1978) In vitro testing of suppressor activity in myasthenia gravis, Muscle and Nerve, 1 : 337. Moretta, L., L. Ferrarini and M. D. Cooper (1978) Characteristics of human T cell subpopulations as defined by specific receptors for immunoglobulins. In : Contemporary Topics in lmmunobiology, Vol. 8, pp. 19-53. Moretta, L., M. Ferrarini, M. C. Mingari, A. Moretta and S. R. Webb (1976) 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. Cool~er (1977) Functional analysis of two human T cell populations - - Help and suppression of B cell responses by T cells bearing receptors for IgM (TM) or IgG (T~), J. exp. Med., 146: 184. Opelz, G., J. Keesey, M. M. Glovsky and R. P. Gale (1978) Autoreactivity between lymphocytes and thymus cells in myasthenia gravis, Arch. Neurol. (Chic'.), 35: 413-415. Penn, A. S., D. L. Schotland and L. P. Rowland (1971) Immunology of muscle disease, Res. Publ. Ass. Res. herr. ment. Dis., 49: 215-240. Piantelli et al. (1979) Subpopulations of T lymphocytes in myasthenia gravis patients, Clin. exp. lmmunol., 36: 85-89. Santoli, D., L. Moretta, R. P. Lisak, D. Gilden and H. Koprowski (1978a) Imbalances in T cell subpopulations in multiple sclerosis patients, J. lrnmunol., 120: 1369-1371. Santoli, D., G. Trinchieri, L. Moretta, C. H. Zmijewski and H. Koprowski (1978b) Spontaneous cell mediated cytotc-xicity in humans - - Distribution and characterisations of the effector cells, Clin. exp. Immunol., 33: 309-318. Shirai, T., M. Miyata, A. Nakase and T. ltoh (1976) Lymphocyte subpopulation in neoplastic and non-neoplastic thymus and in blood of patients with myasthenia gravis, Clin. exp. Immunol., 26: 118-123. Simpson, J. A. (1960) Myasthenia gravis - - A new hypothesis, Scot. reed. J., 5 : 419-435. Simpson, J. A. (1977) Myasthenia gravis - - Validation of a hypothesis, Scot. reed. J., 22: 201-210. Simpson, J. A. (1978) Myasthenia gravis - - A personal view of pathogenesis and mechanism, Part 1, Muscle and Nerve, 1 : 45-56. Simpson, J. A., P. O. Behan and H. Dick (1976) Studies on the nature of autoimmunity in myasthenia gravis - - Evidence for an immuno-deficiency type, Ann. N. Y. Acad. Sci., 274: 382-389. Smiley, J. D., J. Bradley, D. Daly and M, Ziff (1969) Immunoglobulin synthesis in vitro by human thymus - - Comparison of myasthenia gravis and normal thymus, Clin. exp. Immunol., 4: 387 399. Strauss, A. J. I_,.,B. C. Seegal, K. C. Hsu, P. M. Burkholder, W. L. Nastuk and K. E. Osserman (1960) Immunofluorescence demonstration of a muscle binding, complement-fixing serum globulin fraction in myasthenia gravis, Proc. Soc. exp. biol. Med., 105: 184-191. Vincent, A., G. K. Scadding, H. C. Thomas and J. Newson-Davis (1978) In vitro synthesis of antiacetylcholine receptor antibody by thymic lymphocytes in myasthenia gravis, Lancet, 1 : 305-307.