Antibodies against acetylcholine receptor (AChR) can be detected in most patients with myasthenia gravis (MG) and are considered to be involved in the immunopathogenesis of this disease. AChR are isolated from crude receptor preparations by binding to a-bungarotoxin (a-BuTx). Patients with MG have also antibodies against a second protein tentatively named presynaptic membrane receptor (PsmR), which has been isolated from crude receptor utilizing/3-bungarotoxin (/3-BuTx). PsmR could represent another antigen besides AChR relevant for the development of MG. We have now evaluated the T cell reactivity to PsmR in MG and controls by analysing the frequencies of cells which in response to PsmR in short-term cultures secreted interferon-gamma (IFN-y). The B cell response to PsmR was analysed in parallel by counting cells secreting anti-PsmR antibodies. Most patients with MG had PsmR reactive T cells in blood with a median number of about 1 per 44 000 mononuclear cells. Cells secreting anti-PsmR antibodies belonging to the IgG and IgA isotypes, less frequently of the IgM isotype were detected in most MG patients. A positive correlation was found between T cells reactive with PsmR and anti-PsmR IgG antibody secreting cells. PsmR reactive T and B cells were also detected in control patients, but at much lower numbers. Our results indicate that MG is accompanied by T as well as B cell responses to PsmR, in addition to the previously recognized responses to AChR. It remains to be shown whether these PsmR reactivities are of pathogenetic importance in MG.
Introduction
Since 1973 myasthenia gravis (MG) has been consid- ered as an autoimmune disease that results from anti- body-mediated damage of the acetylcholine receptor (AChR) at the neuromuscular junction (Patrick and Lindstr6m 1973). Much evidence has been presented supporting this hypothesis, including the induction of experimental allergic MG (EAMG) by immunization with AChR (Patrick and Lindstr6m 1973)4 This protein
t Dr. Sun is a guest scientist from the Department of Neurology, First Hospital, Harbin Medical University, Harbin, China.
Correspondence to: Dr. H. Link, Department of Neurology, Karolinska Institutet, Huddinge University Hospital, S-141 86 Hud- dinge, Sweden. Tel.: (8)746 54 14; Fax: (8)774 48 22.
binds to a-bungarotoxin (a-BuTx), a phenomenon that is used to isolate AChR from crude receptor prepara- tions (Fambrough et al. 1973). EAMG is also induced by passive transfer of lgG or sera from patients with MG to mouse, or from rats with EAMG to healthy rats (Lennon et al. 1975; Lindstr6m et al. 1976; Toyka et al. 1977). Anti-AChR antibodies acting on the AChR may thereby cause the abnormal muscular fatigue and other signs of MG (Lindstr6m et al. 1976; Lefvert et al. 1978). Anti-AChR antibodies have been detected in serum from 63 to 93% of MG patients (Hinman and Hudson 1983; Tu and Zhang 1985; Vincent and New- som-Davis 1985). There is no clearcut correlation be- tween severity of MG and anti-AChR antibody levels, and patients with generalized MG may be devoid of detectable anti-AChR antibodies in serum (Engel 1986; Newsom-Davis et al. 1987). Additionally, EAMG has
174
also been induced by passive transfer of anti-AChR antibody negative sera from patients with MG (Moss- man et al. 1986; Burges et al. 1987; Drachman et al. 1987). This could indicate immune reactivity to other target structures than AChR in the pathogenesis of MG.
The occurrence of T cell abnormalities in MG is less well defined. One recent study showed T cell reactivity against AChR in only 17% of patients with MG and also in 9% of controls when T cell proliferation in response to AChR was assayed (Newsom-Davis et al. 1989). T cell lines, specific for AChR, have been prop- agated from the blood of MG patients (Hohlfeld et al. 1984), and found to recognize predominantly AChR a subunits (Hohlfeld et al. 1987; Tami et al. 1987) as well as its peptides (Harcourt et al. 1988). When applying the principle of interferon-gamma (IFN-3,) secretion by individual memory T cells after activation with appro- priate antigen (Hecht et al. !o85; Kabilan et al. 1990), we have recently demonstrated high numbers of AChR reactive T cells in blood from most patients with MG, suggesting that autoimmune T cells are regularly in- volved in MG (Link et al. 1991).
We have earlier shown that antibodies directed against a /]-bungarotoxin (/3-BuTx) binding protein occur in MG (Lu et al. 1991). Among 82 Chinese patients with MG, 67% had such antibodies of the IgG isotype in serum. IgG antibodies to /3-BuTx binding protein in the absence of detectable anti-AChR anti- body response were demonstrated in 13% of the pa- tients. This protein, tentatively named presynaptic membrane receptor (PsmR), can be isolated from crude extracts of synaptic membrane fractions by use of its specific binding to/3-BuTx (Lu et al. 1991; Xiao et al. 1991a). SDS polyacrylamide gel electrophoresis of PsmR revealed two peptides with molecular weight of approximately 69 kD and 41 kD. It does not bind to a-BuTx and contamination with AChR was nonde- tectable (Xiao et al. 1991b). PsmR seems thus to repre- sent a second autoantigen in MG and could constitute an additional target for autoimmune attack besides AChR.
We have now examined T and B cell responses to PsmR in both patients with MG and in controls. The presence of T cell reactivity to PsmR was evaluated in an immunospot assay by identifying and enumerating individual cells which secreted IFN-3, after short term culture in re~ponse to PsmR, while the B cell response was evaluated by identification and enumeration of individual cells secreting antibodies against this pro- tein. The T and B cell responses to AChR were also analysed in parallel. Our findings indicate that MG is accompanied by T as well as B cell responses to the /]-BuTx binding protein PsmR, in most cases occuring in parallel with T and B cell responses to AChR.
Materials and methods
Patients Thirty-two patients (24 females) with MG with an
age varying between 29 and 74 years (mean 53) were included. The diagnosis was based on clinical signs and symptoms, previous laboratory tests including serum antibodies to AChR and single fibre EMG abnormali- ties, and response to treatment with anti-cholinesterase inhibitors. Twenty-two patients had been thymec- tomized. The interval between onset of symptoms of MG and the present study was 1-38 years (mean 17 years). Immunosuppressive drugs were administrated to 21 of the patients at the time of blood sampling.
Fourty-six patients (28 females) with other neurolog- ical diseases (OND) were also studied with an age varying between 24 and 80 years (mean 49). Sixteen had tension headache, 15 had multiple sclerosis, 5 had polyneuropathy, 3 had cerebrovascular diseases, 2 had amyotrophic lateral sclerosis, and one patient each had Guillain-Barr~ syndrome, Bell's paresis, varicella-zos- ter mononeuritis, benign intracranial hypertension and head trauma.
Twelve subjects (8 females) were healthy collabora- tors from the Department. Their age was 24-60 years (mean 32).
Preparation of crude receptors Bovine diaphragm muscle was prepared free from
connective tissue and excess of fat, cut into small pieces and stored at -30°C for at most a few weeks, or immediately processed further. 100 g of tissue was homogenized in 400 ml of cooled receptor buffer con- taining 10 mM phosphate buffer saline, pH 7.4 (PBS), 2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride and 100 mM sodium chloride. After centrifugation at 20000 × g for 30 min, the pellet was resuspended in receptor buffer and, again, centrifuged. The pellet was then resuspended in 200 ml of receptor buffer contain- ing 2% Triton X-100 (Sigma, St. Louis, MO, U.S.), stirred for 90 min and subsequently centrifuged at 100000 × g for 1 h. The clear-redish interphase con- taining crude receptor was collected and stored at -70°C until use.
Puripcation of PsmR The /3-BuTx binding protein, PsmR, was purified
from bovine crude receptor preparation (see above) by a series of affinity chromatosraphies on lentil lectin Sepharose 413 (Pharmacia, Uppsala, Sweden),/3-BuTx Affigel 15 (Bio-Rad, Richmond, CA, U.S.) and wheat germ agglutinin Sepharose 6MB (Pharmacia) (Schmidt and Betz 1988; Lu et al. 1991; Xiao et al. 1991a). Purity was assessed by sodium dodecyl sulphate polyacryi- amide gel electrophoresis (SDS-PAGE) and nSl-a-
175
BuTx and ~25i./3.BuTx (Sigma) binding assays. Contam- ination with AChR was < 3%.
Preparation of acetylcholine receptor The a-BuTx binding protein AChR was purified
from bovine diaphragm (Almon et al. 1974). Briefly, following detergent extraction with 1.5% Triton-X-100 and 25% glycerol, and affinity chromatography on lentil lectin-Sepharose 4B column, bound protein was eluted with 0.3 M methyl-2-D-mannopyranoside followed by dialysis against buffer consisting of 1 mM CaCI2 and 5 mM MnCl 2. The isolated fraction was cycled continu- ously through a 2 ml bed volume of a-BuTx-Affigel 15 column (Bio-Rad) at 4°C for 24 h. Bound AChR was eluted with 1 M carbamylcholine chloride followed by dialysis against PBS.
No cross-reactions were found between purified PsmR and AChR, as demonstrated by tzsI-a-BuTx and nsI-/3-BuTx binding.
Preparation of MBP and peripheral nervous system myelin Myelin basic protein (MBP) was isolated from hu-
man brain (Deibler et al. 1972), while peripheral ner- vous system myelin (PNSM) was prepared from bovine lumbosacral plexus (Kadlubowski et al. 1980). The MBP preparation gave one single band on SDS PAG elec- trophoresis.
Preparation of mononuclear cells from peripheral blood Venous blood was collected into heparinized tubes,
and diluted with the same volume of tissue culture medium consisting of Dulbecco's modification of Ea- gle's medium (Flow Lab., Irvine, U.K.) and antibiotics. The peripheral blood mononuclear cells (PBL) were separated by density gradient centrifugation on Lym- phoprep (Nyegaard, Oslo, Norway) for 20 min at 2000 rpm. The PBL from the interphase were carefully collected and washed 3 times with medium and antibi- otics, resuspended in medium supplemented with 2 mmoi L-glutamine (Flow), 1% (v/v) minimal essential medium (Flow), 10% (v/v) fetal calf serum (Gibco, Paisley, U.K.) and antibiotics. The cells were counted and adjusted to 10 6 cells/ml.
Detection of autoreactive T cells The principle of detection of IFN-y secretion by
individual activated T lymphocytes upon recognition of the relevant antigen was used to evaluate presence of antigen-specific memory T cells (Olsson et ai. 1990). Briefly, 96-well microtitre plates and nitrocellulose bot- toms (Millititre-HA, Millipore Co., Bedford, MA, U.S.) were coated with 100 ~1 aliquots of mouse monoclonal anti-human IFN-y antibody (7-B6-5) (Andersson et al. 1989) at a final concentration of 6 /~g/ml at 4°C over night and then washed with PBS. 150/~l of inactivated 5% fetal calf serum in PBS were applied at 37°C for 1
h to block non-specific binding sites on the nitrocellu- lose. 200 /~1 aliquots containing 2 × l0 s PBL were added to individual wells. Antigens in the form of PsmR and AChR were added in 10/~i aliquots to a final concentration of 10/zg/ml. This concentration of antigen gave a maximum number of spots (see below) in preliminary experiments. For each specimen wells receiving no antigen were used as background control ("0 antigen"), wells receiving MBP were used as non- relevant control, and wells receiving purified protein derivate (PPD) (Staten Seruminstitut, Copenhagen, Denmark) and phytohemagglutinin (PHA; Difco, De- troit, MI, U.S.A.)were used as positive controls. After incubation of cultures for 48 h at 370C in a humid atmosphere with 7% CO2, the plates were washed with PBS. To detect IFN-y captured at sites of its secretion, 100/zl aliquots of a rabbit polyclonal anti-human IFN-y antiserum (Interferon Sciences, New Brunswick, N J, U.S.) diluted 1/500 were added to each well for 4 h at room temperature. After washing, biotinylated anti- rabbit IgG (Vector Lab., Burlingame, CA, U.S.) di- luted 1/1000 was added for 2 h, followed by addition of 100 ~1 of avidin-biotin peroxidase complex (ABC Vectastain-Elite Kit; Vector) diluted 1/200. After one hour at room temperature, peroxidase staining was performed (Kaplow 1975). Spots which corresponded to cells that had secreted IFN-y were enumerated with a dissection microscope under 25 × magnification. No spots appeared in specificity control experiments in which the captured antibody was substituted by a mouse anti-rat IFN-y antibody (DB-1; gift from Dr. van der Meide, TNO Primate Center, Rijswijk, The Nether- lands) or an irrelevant mouse monoclonal antibody, or the rabbit polyclonal antibody was omitted. Results are expressed as numbers of spots per l0 s PBL after subtraction of value from "0 antigen" background con- trol culture.
Enumeration of anti-AChR and PsmR antibody secreting cells
A solid-phase enzyme-linked immunospot assay with 96-well microtitre plates and nitrocellulose bottoms (see above) was used as described (Baig et al. 1989; Olsson et al. 1990). The wells were coated with 100 ~! per well of an optimal dilution of PsmR (5 ~g/ml) or AChR (1.25/~g/ml) in PBS. For reference, wells were also coated with PNSM as control antigen (50/zg/ml). These antigen concentrations in the coating solutions were found to be optimal in preliminary experiments for enumeration of antibody secreting cells. After the wells had been coated over night at 4°C, the super- natants were removed by suction through the nitrocel- lulose membranes. The wells were then washed and blocked with 150/~1 aliquots of 5% fetal calf serum at 37°C for one hour. 2 × 105 PBL were added to individ- ual wells. After incubation over night at 370C in humid-
176
ified atmosphere, the wells were emptied, washed, and 100 /.d of diluted high-affinity purified biotinylated goat anti-human IgG, IgA or IgM antiserum (Sigma) were added per well. Washing, incubation with avidin- biotin peroxidase complex, staining and counting of immunospots followed as described above. Values ob- tained were standardized to numbers of spots per 105 cultured cells. Variation of quadruplicate cultures was regularly less than 10%. We have previously docu- mented that the spots which we detected in this system corresponded to secretion of antibodies (Zachau et al. 1989).
Statistical analyses Mann-Whitney's U-test and Spearman's rank corre-
lation test were used for statistical evaluation.
Results
Autoreactive T cells Most patients with MG had in peripheral blood T
cells reactive with the PsmR preparation, as deter- mined by secretion of IFN-3, in response to this anti- gen (Table 1; Fig. 1). The median value of PsmR reactive T cells in the MG patient group was 2.3 per l0 s mononuclear cells, corresponding to 1 per 43,478 cells. The numbers were higher in MG compared to patients with OND (P<0.05) and healthy subjects (P < 0.01). Among the 21 patients with OND, PsmR reactive T cells were detectable in 5 patients. The diagnoses among these 5 patients were multiple sclero- sis in 2 patients, amyotrophic lateral sclerosis in 2 and tension headache in one. Only 2 of the 12 healthy subjects examined had PsmR reactive T cells.
The patients with MG had also higher numbers of AChR-reactive T cells compared to patients with OND
10
y 5
" 6
_w
i - 4
P s m R -2? • 12
e o e o
e e •
ee
10
. 20 • 12
e e
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AChR
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mu ilat m Ism llla an" MG OND H MG OND H
Fig. 1. Numbers of IFN-3, secreting cells per l0 s mononuclear cells from peripheral blood from patients with myasthenia gravis (MG), and from controls consisting of patients with other neurological diseases (OND) and healthy subjects (H), after short-term culture in presence of antigen in the form of presynaptic membrane receptor (PsmR) or acetyicholine receptor (AChR). Values given represent those after subtraction of values obtained from 0 antigen background
control culture (see Methods).
(P < 0.01), and also when compared to healthy subjects ( P < 0.001) (Table 1; Fig. 1). The median value of AChR reactive T cells in the MG patient group was 1.5 per 105 mononuclear cells, corresponding to 1 per 66667 cells. The diagnoses in the OND patients with AChR reactive T cells were tension headache in 2 patients, multiple sclerosis, amyotrophic lateral sclero- sis and polyneuropathy in one patient each.
TABLE 1
NUMBERS OF IFN-y SECRETING CELLS PER l0 s BLOOD MONONUCLEAR CELLS AFTER CULTURE IN ABSENCE OF ANTIGEN (0 ANTIGEN) AND IN THE PRESENCE OF PsmR, AChR, MBP, PPD AND PHA AFTER SUBTRACTION OF NUMBERS OF SPOTS OBTAINED IN 0 ANTIGEN CONTROL CULTURES, IN MYASTHENIA GRAVIS (MG), OTHER NEUROLOGICAL DISEASES (OND), AND HEALTHY SUBJECTS
MG vs. OND NS 0.05 0.01 NS NS NS MG vs. H NS 0.01 0.001 NS NS NS
177
TABLE 2
NUMBERS OF PATIENTS WITH MG SHOWING REACTIVITY TO PRESYNAPTIC MEMBRANE RECEPTOR (PsmR), ACETYL- CHOLINE RECEPTOR (AChR), BOTH ANTIGENS, OR TO NONE OF THEM IN THE IMMUNOSPOT ASSAYS
NUMBERS OF ANTI-PsmR AND ANTI-AChR ANTIBODY SECRETING CELLS OF THE IgG, IgA AND IgM ISOTYPES PER 105 MONONUCLEAR CELLS FROM BLOOD IN PATIENTS WITH MYASTHENIA GRAVIS (MG), OTHER NEUROLOGICAL DISEASES (OND) AND HEALTHY SUBJECTS
For reference, numbers of cells secreting antibodies against peripheral nervous system myelin were also enumerated. ND = not enumerated.
H Range 0-1 0-1 ND 0-2 0-2 ND ND ND ND Median 0 0 0 0 No. pos/exam 5/12 2/12 4/12 5/12
P value MG vs. OND 0.001 0.001 NS 0.05 0.05 NS NS 0.05 NS MG vs. H 0.001 0.001 NS 0.01 0.01 ND ND ND ND
AnU-PsmR Ab c e l s pet 10 s MNC
20 •
16
12 *
8 "
ee l e
l e
4 *
me ~ mine
Anti-AChR Ab cells ; H perlO s MNC
Z 1,22
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eeeee 16"*"
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r " i m r MG OND H MG OND H MG OND H
IgG IgA IgG IgA
Fig. 2. Numbers of anti-PsmR and anti-AChR antibody secreting cells of the IgG and lgA isotype per 105 mononuclear cells isolated from blood from patients with myasthenia gravis (MG), other neurological diseases (OND) and healthy subjects (H).
178
Nine of the 21 patients with MG had both PsmR and AChR reactive T cells, while 2 had only PsmR reactive T cells, and one patient had only AChR reac- tive T cells (Table 2). The remaining 9 MG patients had no detectable T cell response to PsmR or AChR. We observed a positive correlation in our MG patients between numbers of PsmR and AChR reactive T cells (r = 0.44; P < 0.05).
Two of the patients with OND (one with multiple sclerosis and one with amyotrophic lateral sclerosis) had T cells responding to both antigens.
When we for reference cultivated PBL in the pres- ence of MBP, PPD, or PHA, we observed no differ- ences for numbers of IFN-~" secreting cells in patients with MG compared to OND or healthy subjects (Table 1). Similarly, no differences were encountered when PBL were cultivated in absence of antigen or lectin ("0 antigen"). These data argue against any general or non-specific T cell hyperreactivity in MG patients' pe- ripheral blood.
Autoantibody secreting cells Cells secreting anti-PsmR antibodies of the IgG
isotype were found in blood from most patients with MG at a median value of 3.8 per 105 PBL, correspond- ing to 1 per 26,316 cells (Table 3; Fig. 2). Such cells were also detected in OND and in healthy subjects, but at lower numbers (P < 0.001). Anti-PsmR IgA and IgM antibody secreting cells were also detected in the MG patients' blood, but less frequently and at lower num- bers compared to IgG antibodies (Table 3). Numbers of anti-PsmR IgA antibody secreting cells were higher in MG compared to OND (P < 0.05) and healthy sub- jects (P < 0.01), while the difference between MG and OND for IgM antibody secreting cells did not reach significance.
A majority of the MG patients had also anti-AChR IgG antibody secreting cells in blood, at a median value of 2.6 per 105 mononuclear cells, corresponding to 1 per 38462 cells (Table 3; Fig. 2). Numbers were again higher in MG compared to OND and healthy subjects (P < 0.001). Most of the MG patients had also anti-AChR antibody secreting cells of the IgA and IgM isotypes, but again less frequently and at lower num- bers compared to anti-AChR IgG antibody secreting cells. Anti-AChR IgA antibody secreting cells were also found in the patients with OND and in healthy subjects, and anti-AChR IgM antibody secreting cells in OND, but at significantly lower numbers compared to MG.
Of the 32 MG patients examined, 21 (66%) had a B cell response to both PsmR and AChR (Table 2). Some patients (16%) had only or mainly cells secreting anti- bodies to PsmR, and some patients (9%) had only or mainly cells secreting anti-AChR antibodies. No cells
secreting antibodies of these two specificities were detected in the few remaining patients.
Among the 11 MG patients examined for cells se- creting anti-PsmR antibodies of all three isotypes, 2 had cells secreting IgG + IgA + IgM, 2 had IgG + IgA, one had IgG + IgM, one had IgA + IgM, and 5 pa- tients had IgG only. Among the 11 MG patients simi- larly examined for cells secreting anti-AChR antibodies of all three isotypes, 3 patients had IgG + IgA + IgM, 2 had IgG + IgA, one had lgG + IgM, 2 had IgG only, 2 had lgM only and one had lgA only.
For reference, 16 of the MG patients and 13 with OND were examined for cells secreting antibodies against peripheral nervous system myelin. Only low numbers of antibody secreting cells were detected, without differences between the groups (Table 3).
Relation between autoreactive T and B cells Positive correlations were found between PsmR re-
active T cells and anti-PsmR IgG antibody secreting cells ( r - 0.51; P < 0.02), PsmR reactive T cells and anti-PsmR IgA antibody secreting cells (r = 0.66; P < 0.002), AChR reactive T cells and anti-AChR IgG antibody secreting cells (r = 0.59; P < 0.005), and be- tween AChR reactive T cells and anti-AChR IgA anti- body secreting cells (r - 0.59; P < 0.005).
Twenty-two of the patients with MG had been thymectomized. The mean values for PsmR and AChR reactive T and B cells did not differ when compared with those patients who had not been subjected to thymectomy (data not shown).
Discussion
Utilizing an immunospot assay for identification of IFN-3, secretion by individual memory T cells after short term culture of blood mononuclear ceils in the presence of the /3-BuTx binding protein PsmR, we have demonstrated that a majority of patients with MG have PsmR reactive T cells. Such cells were also de- tected in peripheral blood from control subjects, al- though less frequently and at lower numbers. Further- more, a majority of the MG patients had also anti-PsmR antibody secreting cells. This B cell response, which mostly involved IgG antibodies and less frequently IgA and IgM antibodies, was also detectable in control subjects, but again less frequently and at lower num- bers. The T and B cell responses to PsmR coincided in most of the MG patients with corresponding T and B cell responses to AChR.
We used PPD and PHA stimulation as positive controls to evaluate specific T cell memory to the common antigen BCG and polyclonal T cell activation, respectively, as promotors to IFN-~ secretion. Based
on the results from these control studies, it is most probable that our findings obtained in presence of PsmR - as well as with AChR - do not reflect any generally increased capacity to produce IFN-7 in re- sponse to antigen or lectin in MG. This conclusion is further strengthened by our results with MBP, which is an antigen unrelated to MG but of possible relevance as target for autoimmune attack in multiple sclerosis (Olsson et al. 1990). No differences between MG and our two control groups were detected for MBP reactive T cells.
AChR specific T cell responses in human MG have mostly been evaluated by T cell proliferation assays using [3H]thymidine incorporation (Abramsky et al. 1975; Newsom-Davis et al. 1989), and by establishing T cell lines to AChR (Hohlfeld et al. 1984) or to AChR subunits (Brocke et al. 1988; Newsom-Davis et al. 1989; Protti et al. 1990). With these approaches, it has not been possible to define the antigen specific T cell repertoire in the individual subjects. The numbers of AChR reactive T cells in blood from animals with EAMG are small (DeBaets et ai. 1982), as they are in human MG (Link et al. 1991) - in fact, the median number of AChR reactive T cells in our 21 MG pa- tients was only 1 per 67 000 mononuclear blood cells - and this could be one reason why the previously used assays have been difficult to interprete in terms of T cell repertoire in the individual subject. We have also recently shown that lymphocyte proliferation assays reveal AChR T cell reactivities much less frequently in comparison with the T cell immunospot assay used in the present study (Sun et al., submitted). With this assay that allows both iJentification and enumeration of antigen specific memory T cells, we now found that a substantial proportion of the patients with MG had T cells recognizing PsmR and AChR in parallel, while a few patients had T cells recognizing one of these two antigens, and another subgroup was devoid of PsmR and AChR reactive T cells in blood. Still, the numbers which we detected represent only minimum frequen- cies, and T cells which upon activation secrete IL-4, i.e. TH2 cells, are not taken into account (Mossman & Coffman 1989).
PsmR and/or AChR reactive T cells were also detected in control patients but less frequently and at lower numbers compared to MG. The occurrence of AChR reactive T cells in controls is in agreement with previous observations from proliferation assays and results from propagation of AChR reactive T cell lines. It could well be that such T cells represent a fraction of a naturally occurring T cell repertoire. The different ways by which tolerance to AChR can be broken, giving rise to an abnormal T cell dependent response to AChR, has recently been reviewed (Steinman and Mantegazza 1990). Thus, AChR reactive T cells could be tolerated if muscle cells do not express MHC class
179
II, or B cells which constitutively express MHC class II, do not recognize AChR and, in analogy, the /3-BuTx binding protein PsmR.
The cytokine IFN-7 which is secreted by memory T cells after activation, has multiple effects, including activation of macrophages, induction of T cell homing and MHC antigens. All these effects could be impor- tant in the induction and persistence of an inflamma- tory response at the target for autoimmune attack. The T cell immunospot assay based on identification of IFN-~, secretion has thus the additional advantage that it rests on the demonstration of an effector molecule with possible pathogenetic relevance. This assay should be useful for further definition of presence and patho- genetic importance of T cell responses to subunits of AChR, peptides of such subunits, and components of PsmR.
Utilizing the principle of detection of antibodies secreted by individual cells, we have now confirmed the previous observation that the B cell response directed against AChR in MG patients involves predominantly IgG antibodies, but IgA and IgM antibodies as well (Link et al. 1991). These specific lgA and IgM antibody responses have previously not been detected, probably because of the lower sensitivity inherent to determina- tions of antibody levels in body fluids due to e.g. binding of measurable antibodies to muscle in vivo and short half life of autoantibodies (Lefvert 1978; Link et al. 1988). The B cell response to PsmR in patients with MG is also predominated by antibodies of the IgO isotype, but anti-PsmR antibodies of IgA and IgM isotypes may occur as well.
The occurrence of specific antibody secreting cells in control patients is in agreement with our previous report of anti-AChR antibody secreting cells in a sub- stantial proportion of non-MO patients (Link et al. 1991). Both anti-AChR and anti-PsmR antibodies could represent natural antibodies. Due to a mechanism which remains unknown, an expansion of B cells with specificities for components of the neuromuscular junction is induced in MG, participating in the destruc- tive events in MG. Alternatively, the high production of anti-AChR and anti-PsmR antibodies in MG could be secondary to lesions of the neuromuscular junction. These two possibilities are not mutually exclusive. Molecules that mimic AChR have been described in e.g. gram-negative bacteria and herpes simplex virus (Stefanson et al. 1986; Schwimbeck et al. 1989). Infec- tion with such microorganisms and development of cross-reactive antibodies could then be another expla- nation for occurrence of AChR reactivities in both MG and non-MG subjects.
The present detection of PsmR reactive T cells, as well as of anti-PsmR antibody secreting cells, in most MG patients is in line with the hypothesis that MG could be due to immune damage not only of the
180
AChR, but concordantly a consequence of immune- mediated damage of /3-BuTx binding sites (Lu et al. 1991).
Acknowledgements This study was supported by grants from funds from Karolinska lnstitutet, the Swedish Multiple Sclerosis Society (NHR) and the Swedish Medical Research Council. We thank Dr. Georg Matell, S6dersjukhuset, Stockholm, for permission to include his patients in this study and for valuab|e c,~mmen~s regarding the manuscript, and Ms. Yvonne Nilsson for ex,:ellent secretarial help. We thank Gudrun Andersson, Department of Clinical Immunology, University of Lurid, for the generous gift of antibody 7-B6-1.
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