Autoreactive T cells to the P3A+ isoform of AChR α subunit in myasthenia gravis

Journal of Neuroimmunology 137 (2003) 177 – 186 www.elsevier.com/locate/jneuroim

Autoreactive T cells to the P3A+ isoform of AChR a subunit in myasthenia gravis Shigeaki Suzuki a, Kortaro Tanaka a, Hidekata Yasuoka b, Yasuo Fukuuchi a, Yutaka Kawakami c, Masataka Kuwana c,* a Department of Neurology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan c Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan b

Received 24 June 2002; received in revised form 4 December 2002; accepted 12 February 2003

Abstract In vitro T cell proliferative response to an alternative splicing variant of acetylcholine receptor a subunit (AChRa) with the P3A exonencoded region was examined in peripheral blood samples from 28 myasthenia gravis (MG) patients and 14 healthy donors using recombinant fragments and synthetic peptides. T cells responsive to the P3A region-specific sequences were detected in five MG patients, all of whom were late-onset disease with thymoma, but in none of healthy donors. These autoreactive T cells may be involved in the pathogenic process in a subset of MG patients. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Acetylcholine receptor a subunit; P3A exon; T cell; Myasthenia gravis; Thymoma

1. Introduction Myasthenia gravis (MG) is an organ-specific autoimmune disorder mediated by autoantibodies to the nicotinic acetylcholine receptor (AChR) (Drachman, 1994). AChR is a glycoprotein having a molecular weight of approximately 250 kDa that projects through the muscle membrane and is composed of five subunits, arranged like barrel staves around a central channel. The adult form of AChR is composed of two a subunits and one of h, y, and q subunit each. The q subunit is replaced by a g subunit in fetal and denervated muscle (Drachman, 1994). The a subunit of AChR (AChRa) contains both the site for acetylcholine binding and the main epitopes recognized by autoreactive T cells and autoantibodies in MG patients (Hawke et al., 1996; Conti-Fine et al., 1998). There are two isoforms of AChRa, P3A+ and P3A , as a result of alternative splicing. The P3A+ isoform of AChRa has an additional 25-amino acid insert encoded by the P3A exon between amino acid (AA)

* Corresponding author. Tel.: +81-3-5363-3778; fax: +81-3-53629259. E-mail address: [email protected] (M. Kuwana).

positions 58 and 59 of the P3A isoform (Beeson et al., 1990). The P3A isoform has been shown to be a functional subunit based on electrophysiologic analysis, but a physiological role of the P3A+ isoform remains unclear (Newland et al., 1995). Expression of both P3A+ and P3A isoforms are detected in skeletal muscle and thymus (MacLennan et al., 1993; Andreetta et al., 1997). Guyon et al. (1994) reported that expression levels for mRNAs encoding the P3A+ and P3A isoforms were almost equal in skeletal muscle obtained from both MG patients and healthy individuals. The mRNA expression of the P3A+ isoform was subsequently shown to be upregulated in abnormal thymus, including thymoma and thymic hyperplasia, in MG patients (Wakkach et al., 1996; Zheng et al., 1999; Wilisch et al., 1999), whereas Andreetta et al. (1997) failed to detect the P3A+ isoform in all thymoma examined. Taken together, these reports suggest a possible relationship between overexpression of the P3A+ isoform in abnormal thymus and the autoimmune response to AChR in MG patients. There is accumulating evidence that autoreactive T cells to AChRa are involved in the production of pathogenic anti-AChR antibodies in MG patients (Hawke et al., 1996; Conti-Fine et al., 1998). However, most studies have utilized synthetic peptides or recombinant proteins correspond-

0165-5728/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0165-5728(03)00078-X

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ing to the protein sequence of the P3A isoform (Brocke et al., 1988; Oshima et al., 1990; Manfredi et al., 1994). In fact, the majority of T cell epitopes identified in the previous reports were mapped on the P3A isoform (Hawke et al., 1996; Conti-Fine et al., 1998; Brocke et al., 1988; Oshima et al., 1990; Manfredi et al., 1994), and only limited information regarding T cell responses to the sequences encoded by the P3A exon is available to date (Harcourt et al., 1993; Matsuo et al., 1995; Kanai et al., 1997). In this study, autoreactive T cell response to the P3A+ isoform of AChRa was examined in MG patients and healthy individuals by using soluble recombinant fragments and synthetic peptides encoding the P3A-encoded region. The clinical characteristics of the MG patients with a T cell response specific to the P3A-encoded region were also evaluated.

2. Patients and methods 2.1. Patients and controls Peripheral blood samples from 28 MG patients (MG1– 28; 14 males and 14 females) and 14 healthy donors (HD1 –14; 7 males and 7 females) were analyzed in this study. The diagnosis of MG was based on clinical, electrophysiologic, and immunologic criteria (Drachman, 1994). The mean age of the MG patients at blood collection was higher than that of the healthy donors (56.9 F 17.8 versus 34.7 F 9.8 years; p < 0.001). The mean disease duration of MG at blood specimen collection was 8.7 F 5.5 years. There are 6 patients that were diagnosed with ocular MG, and the other 22 patients had generalized MG. The MG severity was graded according to the Task Force of the Medical Advisory Board of the Myasthenia Gravis Foundation of America (Jaretzki et al., 2000). Trans-sternal extended thymectomy was performed in 17 MG patients, and histopathologic examination revealed 2 with normal thymus, 6 with thymic hyperplasia, and 9 with thymoma. Serum anti-AChR antibody levels were measured by a conventional radioimmuno-precipitation assay (normal range: < 0.2 nM) (Drachman, 1994), and the maximum level in each MG patient was recorded. Twenty-four patients (86%) were positive for anti-AChR antibodies at least once during the course of disease (range: 0.4 – 750 nM). At the time of the blood examination, the disease was generally in remission: 11 patients were taking low-dose corticosteroids, and the mean daily dosage of prednisolone was 5.7 mg (range: 2– 10 mg). All blood specimens were collected after the patients and controls gave their written informed consent approved by the Keio University Institutional Boards. 2.2. HLA class II allele genotyping Genomic DNA was extracted from peripheral blood nucleated cells and subjected to the polymerase chain

reaction (PCR) using primers specific for the second exon of the DRB1 or DQB1 gene. HLA class II alleles were determined based on the restriction fragment length polymorphisms of the PCR-amplified products (Inoko and Ota, 1993). 2.3. Preparation of recombinant AChRa fragments and synthetic peptides Recombinant maltose-binding protein (MalBP) fusion proteins expressing the extracellular domain of two AChRa isoforms, one containing the P3A-encoded region (rP3A+: amino acid residues AA1 – 58/1+ – 25+/59 – 236) and the other lacking it (rP3A : amino acid residues AA1– 236), were generated using a bacterial expression system as described previously (Kuwana et al., 1995a). Briefly, human rabdomyosarcoma cell line TE671 was treated with 5 AM dexamethasone and 1 mM nicotine to upregulate AChR gene expression (Luther et al., 1989), and total RNA was extracted from the treated cells with an RNeasy Mini kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. A first-strand cDNA was reverse-transcribed from RNA with oligo-dt priming and subjected to PCR using a specific primer pair (forward, 5VTCGTCCTGGGCTCCGAACATGAGA-3V; reverse, 5VGAGTCTGTGGGCAGGTAGAATACC-3V) to amplify DNA covering the entire extracellular domain of human AChRa (Noda et al., 1983). Two DNA fragments, one with and one without the P3A transcript, were subcloned in frame into 3Vend of the MalBP gene in the pMAL-c2 expression vector (New England Biolabs, Beverly, MA). To eliminate possible PCR errors and to verify the translation frames, both strands of each DNA construct were sequenced on an ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA) using the BigDye terminator (Applied Biosystems). Expression of rP3A+, rP3A , and MalBP was induced with isopropyl-h-D-thiogalactopyranoside. These recombinant fragments were purified by amylose-resin affinity chromatography (Maina et al., 1988). To minimize contamination of bacterial components into purified preparations, amylose-resins bound to the MalBP fusion protein were extensively washed with an excess volume of the buffer in a high flow rate, which resulted in decrease of the yield, but greatly reduced contaminating bacterial proteins. The purified preparations were dialyzed against phosphate-buffered saline and filter-sterilized. All batches were mixed together and used in T cell stimulation to prevent difference in stimulating capacity which may vary from batch to batch. The purified proteins (1 Ag/lane) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 5 – 20% polyacrylamide gradient gels, followed by Coomassie blue staining. To examine the degree of contaminating bacterial proteins into purified preparations, an excess amount of each preparation (10 Ag/lane) was also loaded on the gel. Quantitative densitometric analysis of the protein

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bands on Coomassie blue-stained gels was carried out using Molecular Imager FX (Bio-Rad Laboratories, Hercules, CA). rP3A+, rP3A , and MalBP (1 Ag/lane) were also subjected to immunoblot analysis probed with anti-MalBP polyclonal antibodies (New England Biolabs) and monoclonal antibody (mAb) to the extracellular portion of AChRa (Clone D6; Oncogene Research Products, Cambridge, MA). Clone D6 reacts with human AChRa but binds weakly to a synthetic peptide encoding an epitope located at AA64– 78 of AChRa. Nine 15-mer peptides with 10 residue overlaps covering the entire amino acid sequence encoded by the P3A exon (p1, AA44 – 58; p2, AA49 –58/1+ –5+; p3, AA54 –58/1+ – 10+; p4, AA1+ –15+; p5, AA6+ – 20+; p6, AA11+ – 25+; p7, AA16+ – 25+/59 – 63; p8, AA20+ –25+/59 –68; p9, AA59 – 73) were synthesized with solid-phase multiple peptide synthesizer (Advanced Chemtech, Louisville, KY). The purity of all peptide preparations was >50% as determined by high-performance liquid chromatography.

proliferation was expressed as the stimulation index (SI), calculated as the cpm incorporated into rP3A+- or rP3A stimulated cultures divided by the cpm incorporated into cultures stimulated with MalBP, which is presumed to contain bacterial contaminants similar to rP3A+ and rP3A preparations. A positive response was defined as having both a SI>3 and a >500 cpm increase associated with antigen stimulation (Kuwana et al., 1998). In addition, MalBP-specific T cell response was calculated as the cpm incorporated into MalBP-stimulated cultures divided by the cpm incorporated into cultures with medium alone. In some instances, the ratio of the T cell response to rP3A+ and rP3A (rP3A+/rP3A ratio) was calculated as the cpm incorporated into rP3A+-stimulated cultures divided by the cpm incorporated into rP3A -stimulated cultures at an antigen concentration of 10 Ag/ml.

2.4. Cell preparation

T cell lines responsive to rP3A+ were generated by repeated stimulation of PBMCs with rP3A+ as previously described (Kuwana et al., 1997) with some modifications. Briefly, PBMCs (2  106) were cultured with rP3A+ (10 Ag/ ml) in 24-well plates, and interleukin (IL)-2 (30 units/ml; Biogen, Cambridge, MA) was added to the culture on day 3. On days 10 and 17, the cells were restimulated with rP3A+ (10 Ag/ml), IL-2 (100 units/ml), and 2  106 irradiated (40 Gy) autologous PBMCs. On day 24, rP3A+-stimulated T cell lines (5  104) were cultured with rP3A+, MalBP (10 Ag/ml), or a series of the P3A peptides (1 Ag/ml), in the presence of IL-2 (30 units/ml) and 105 irradiated autologous PBMCs in 96-well round-bottomed culture plates for 3 days. Culture supernatants were collected, and the interferon (IFN)-g concentration was measured by enzyme-linked immunosorbent assay as previously described (Kawakami et al., 1994). Optical density at 450 nm was measured, and the IFN-g concentration was calculated in comparison to serial concentrations of recombinant IFN-g (Endogen, Woburn, MA) measured in the same plate. All experiments were carried out in duplicate, and all values are the means of duplicate determinations.

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by density-gradient centrifugation with Lymphoprep (Nycomed Pharma, Oslo, Norway) and cultured in RPMI1640 containing 10% fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin, and 50 Ag/ ml streptomycin at 37 jC, in 5% CO2. In some experiments, CD4+ or CD8+ cell-depleted PBMCs were prepared by incubating PBMCs with anti-CD4 or anti-CD8 mAbcoupled magnetic beads (Dynal, Oslo, Norway), respectively, followed by magnetic removal of bead-bound cells (Kuwana et al., 1995b). 2.5. T cell proliferation assays In vitro antigen-induced T cell proliferation was measured in bulk PBMC cultures as previously described (Kuwana et al., 1995b) with some modifications. Briefly, PBMCs (105) were cultured with or without antigen in 96well flat-bottomed culture plates for 7 days. CD4+ or CD8+ cell-depleted PBMCs were also used in some experiments. During the final 18 h of culture, the cells were incubated with 0.5 ACi/well of 3H-thymidine, and after harvesting, 3Hthymidine incorporation was determined in a Top-Count microplate scintillation counter (Packard, Meriden, CT). Three different concentrations of rP3A+, rP3A , and MalBP (1, 5, and 10 Ag/ml) were used as the antigens for all subjects, while lower antigen concentrations (0.1, 0.2, and 0.5 Ag/ml) were also tested in four MG responders. All cultures were carried out in quadruplicate, and all values are means of quadruplicate determinations [SDs are < 20% of the mean or < 100 counts per minute (cpm), unless otherwise indicated]. To minimize the effect of background T cell response to bacterial components contaminating into rP3A+ and rP3A preparations, rP3A+- and rP3A -specific T cell

2.6. Detection of T cells responsive to the P3A-encoded region

2.7. Blocking of T cell responses by anti-HLA class II mAbs To examine the effects of anti-HLA class II mAbs on antigen-induced T cell proliferation and IFN-g production, anti-HLA-DR, anti-HLA-DQ, and isotype-matched control mAbs (Leinco, St. Louis, MO) were added at a final concentration of 1 Ag/ml at the start of culture (Kuwana et al., 1995b). 2.8. Statistical analysis Statistical comparisons were performed using Fisher’s two-tailed exact test or Student’s t-test. The correlation

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between T cell proliferative responses induced by rP3A+ and rP3A was tested using a single regression model. In some associations with HLA class II antigens, corrected p values were obtained by multiplying observed p values by the number of comparisons made.

3. Results 3.1. Preparation of recombinant AChRa fusion proteins Two recombinant fusion proteins encompassing the entire extracellular domain of AChRa, one with and one without the P3A-encoded region, were successfully expressed and purified. The purified preparations of rP3A+, rP3A , and MalBP were fractionated in a regular condition (1 Ag/lane) and an overloaded condition (10 Ag/ lane), and then stained with Coomassie blue (Fig. 1). A dominant protein band having a molecular weight of approximately 68 kDa (rP3A+), 66 kDa (rP3A ), and several smaller proteins ranging from 50 to 68 kDa were detected in the rP3A+ and rP3A preparations, but other protein bands were scarcely found even in the overloaded condition (10 Ag/lane). The relative amounts of these major protein bands represented >95% of the total stained proteins by densitometry. To test whether these smaller proteins were either degradation products or contaminating bacterialdelivered proteins, MalBP, rP3A+, and rP3A were fractionated on the gels at a dose of 1 Ag/lane and probed with anti-MalBP and anti-AChRa antibodies (Fig. 1). The majority of proteins ranging from 50 to 68 kDa were recognized by anti-MalBP antibodies, suggesting that these protein bands mainly corresponded to intact and degraded recombi-

Fig. 1. Affinity-purified recombinant acetylcholine receptor (AChR) a subunit fusion proteins, with and without the P3A-encoded region, used for T cell stimulation. Purified maltose-binding protein (MalBP), rP3A+, and rP3A (1 or 10 Ag/lane) were applied to sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and the gel was stained with Coomassie blue. In addition, MalBP, rP3A+, and rP3A (1 Ag/lane) were fractionated on the gels and probed with anti-MalBP polyclonal antibody or antiAChRa monoclonal antibody. Mw makers—molecular weight standards.

nant AChRa fragments. rP3A+ and rP3A either in intact or degraded form were also recognized by anti-AChRa mAb, but the intensity of the rP3A+ band was consistently more prominent than the intensity of the rP3A band. Since the anti-AChRa mAb used recognizes the epitope located close to the insertion site for the P3A-encoded sequence, our finding might reflect difference in the binding affinity of the mAb to rP3A+ and rP3A . The antigen preparations at 1, 5, and 10 Ag/ml were confirmed to be nontoxic to T cells because phytohemagglutinin-induced proliferation of peripheral blood T cells was not suppressed in the presence of these preparations (data not shown). 3.2. T cell proliferative response to recombinant AChRa fragments T cell proliferative responses to serial concentrations of rP3A+ and rP3A were examined in 28 MG patients and 14 healthy controls. As shown in Fig. 2, rP3A+ and rP3A induced a T cell proliferative response in the majority of MG patients and healthy controls. Frequencies of responders to rP3A+ and rP3A at different antigen concentrations in MG patients and healthy donors are summarized in Table 1. In MG patients, the frequencies of responders were similar at three different antigen concentrations, and the SI levels for rP3A+- or rP3A -induced T cell proliferation were also similar at antigen concentrations of 1, 5, and 10 Ag/ml. The majority of MG patients who responded to rP3A+/ rP3A at 1 Ag/ml remained responders at 5 and 10 Ag/ml, although some variations in the SI levels were observed. Nonresponders at 1 Ag/ml failed to respond to rP3A+/rP3A at 5 and 10 Ag/ml in most cases, but showed a significant response at higher concentrations of rP3A+ and rP3A in one and three patients, respectively. When T cell responses to lower concentrations of rP3A+ and rP3A were tested in four MG responders, a clear dose-dependent response was detected (Fig. 3), indicating that T cell responses to rP3A+ and rP3A were already saturated at 1 Ag/ml in MG patients. In contrast, a clear dose-dependent response at antigen concentrations ranging from 1 to 10 Ag/ml was detected in healthy donors. In fact, the responses of healthy donors to rP3A+ and rP3A at 10 Ag/ml were significantly higher than at 1 Ag/ml (4.5 F 1.8 versus 2.5 F 0.8, p = 0.004 and 4.3 F 2.4 versus 2.0 F 0.8, p = 0.001, respectively). Of four MG patients negative for anti-AChR antibody, three were responders to both rP3A+ and rP3A . On the other hand, T cells from all MG patients and healthy donors failed to respond to MalBP, which is presumed to contain bacterial contaminants similar to rP3A+ and rP3A preparations. 3.3. Characterization of T cells responsive to rP3A+ The effects of CD4+ or CD8+ cell depletion as well as anti-HLA class II mAbs on rP3A+-induced T cell proliferation in bulk PBMC cultures were examined in the 18

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Fig. 2. T cell proliferative responses to serial concentrations of rP3A+ and rP3A in 28 MG patients and 14 healthy donors. Peripheral blood mononuclear cells were cultured with MalBP, rP3A+, or rP3A (1, 5, and 10 Ag/ml) for 7 days, and 3H-thymidine incorporation was measured by liquid scintillation counting. The stimulation index (SI) was calculated as the cpm incorporated into rP3A+- and rP3A -stimulated cultures divided by the cpm incorporated into MalBPstimulated cultures. The SI level of MalBP was calculated as the cpm incorporated into MalBP-stimulated cultures divided by the cpm incorporated into no antigen. Closed circles and open circles indicate MG patients positive and negative for anti-AChR antibody, respectively. The dotted line marks the cutoff level for a positive response.

rP3A+ responders, who consisted of 12 MG patients and 6 healthy donors. As shown in Fig. 4, rP3A+-induced T cell proliferation was almost completely lost when CD4+ cells, but not CD8+ cells, were depleted in representative subjects. Concordant results were detected in all MG patients and healthy donors examined, indicating that the T cells reactive with rP3A+ were almost exclusively CD4+ T cells. The blocking experiments using anti-HLA-DR and antiHLA-DQ mAbs showed that anti-HLA-DR mAb was highly effective in inhibiting rP3A+-induced T cell proliferation in two MG patients and a healthy donor (Fig. 4). Anti-HLADQ mAb also partially inhibited the T cell response. A

predominant HLA-DR restriction with less contribution by HLA-DQ was detected in all MG patients and healthy donors. 3.4. Comparison between rP3A+- and rP3A -induced T cell responses The SI levels for rP3A+- and rP3A -induced T cell proliferation at an antigen concentration of 10 Ag/ml were significantly correlated with each other in both MG patients and healthy donors (r = 0.84, p < 10 6 and r = 0.42, p = 0.01, respectively), but there was a trend toward a preferential T

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Table 1 Positive response rates of T cell proliferation to serial concentrations of rP3A+ and rP3A in MG patients and healthy donors Antigen (Ag/ml) rP3A+ 1 5 10 rP3A 1 5 10

MG patients (n = 28) (%)

Healthy donors (n = 14) (%)

57 61 61

36 43 71

39 50 50

14 43 71

MG6

MG25

HD10

A positive response was defined as having both a stimulation index (SI) >3 and >500 cpm increase associated with antigen stimulation.

cell response to rP3A+ in MG patients, and not in healthy donors (Fig. 5). However, the rP3A+/rP3A ratios in MG patients and healthy donors were not statistically different (1.24 F 0.55 versus 1.13 F 0.42, p = 0.28). To identify a subset of MG patients who preferentially responded to rP3A+, the rP3A+/rP3A ratio was compared in the presence or absence of various clinical findings and individual HLA class II alleles. The results revealed that the rP3A+/ rP3A ratio was significantly higher in 14 MG patients with DQ4 (DQB1*0401/*0402) compared to 14 MG patients without (1.52 F 0.43 versus 1.08 F 0.48, corrected p value = 0.02). In contrast, the rP3A+/rP3A ratio was not different between 10 healthy controls with DQ4 and 4 without DQ4 (0.98 F 0.33 versus 1.05 F 0.43). These findings strongly suggest the presence of T cells responsive to the rP3A+-specific sequences in a subset of MG patients, especially those with DQ4. 3.5. Detection of T cells responsive to the P3A-encoded sequences

Fig. 4. Effects of CD4+- or CD8+ cell depletion and anti-HLA class II mAb on T cell proliferation induced by rP3A+ in MG patients (MG6 and MG25) and a healthy donor (HD10). Peripheral blood mononuclear cells that were untreated, CD4+ cell-depleted, or CD8+ cell-depleted were cultured with rP3A+ for 7 days, and 3H-thymidine incorporation was measured by liquid scintillation counting. Anti-HLA-DR, anti-HLA-DQ, and isotype-matched control mAbs were added at the start of the cultures. SDs are not shown when results are < 20% of the mean or < 100 cpm.

responders consisting of 14 MG patients and 10 healthy donors. The results showed that T cell responses to any of the P3A peptides were detected in 5 of 14 MG patients,

To detect T cells responsive to the P3A-encoded sequences, peripheral blood T cells were stimulated repeatedly with rP3A+ and examined for their ability to produce IFN-g in response to a series of P3A peptides in 24 P3A+

Fig. 3. T cell proliferative responses to lower antigen concentrations of rP3A+ and rP3A in four MG responders. The stimulation index (SI) was calculated as the cpm incorporated into rP3A+- and rP3A -stimulated cultures divided by the cpm incorporated into MalBP-stimulated cultures. The dotted line marks the cutoff level for a positive response. See Fig. 2 for other definitions.

Fig. 5. Correlation between stimulation index (SI) levels for rP3A+- and rP3A -induced T cell proliferation in 28 MG patients and 14 healthy donors. SI was calculated at an antigen concentration of 10 Ag/ml. Open diamonds and closed diamonds indicate MG patients and healthy donors with and without DQ4, respectively. Dotted line represents equal SI levels for rP3A+- and rP3A -induced T cell responses (rP3A+/rP3A ratio = 1). See Fig. 2 for other definitions.

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Fig. 6. T cell responses to overlapping synthetic peptides covering the P3A-encoded region (p1 – p9) in two representative MG patients. Interferon-g (IFN- g) production of rP3A+-stimulated T cell lines in response to antigenic stimulation was measured by enzyme-linked immunosorbent assay. A significant IFN-g level in cultures with the peptide compared with the cultures without the peptide is marked by an asterisk. SDs are not shown when results are < 20% of the mean or < 20 pg/ml. The numbers on the AChRa sequence indicate the amino acid positions. Representative results of two independent experiments are shown.

but in none of 10 healthy donors ( p = 0.053). Representative results of the peptide-induced IFN-g production in rP3A+-stimulated T cell lines generated from two MG patients are shown in Fig. 6. Similar findings were reproduced and obtained by repeated examination using PBMC samples obtained at independent time points. The rP3A+-stimulated T cells generated from MG15 produced IFN-g in response to p4, p5, and p6, whereas those from MG21 responded to p6 and p7. The antigenic peptides, clinical features, and HLA class II alleles of the five MG patients who responded to the P3A peptides are summarized in Table 2. The patients could be divided into two

groups on the basis of the immunoreactive peptides: three patients (MG6, 11, and 15) who responded to the center of the P3A-encoded region (p5, AA6+ –20+); and two patients (MG7 and 21) who responded to the border of the carboxyl-terminus of the P3A-encoded region and the subsequent shared sequence (p7, AA16+ – 25+/59 – 63). Three MG patients with p5-reactive T cells possessed DQB1*0402 in common, whereas two patients with p7reactive T cells had DQB1*0601. The blocking experiments with anti-HLA class II mAbs revealed that antiHLA-DQ mAb selectively inhibited the peptide-induced IFN-g synthesis in MG7 and MG15 (data not shown). As

Table 2 Clinical characteristics, HLA class II alleles, and antigenic peptides of MG patients who showed a T cell response specific to the P3A-encoded region

MG6 MG11 MG15 MG7 MG21

Age/ gender

Age at disease onset

MG gradea

Thymus histology

Anti-AChR antibody titer (nM)

HLA class II alleles DRB1

DQB1

61/M 71/F 66/M 62/M 68/F

46 63 62 46 49

IIIb IIa V IIIb I

thymoma invasive thymoma invasive thymoma invasive thymoma thymoma

14 1.6 66 8.7 29

*1502/*0410 *1502/*0410 *1102/*0802 *0404/*0803 *1502/*1201

*0602/*0402 *0601/*0402 *0303/*0402 *0601/*0302 *0601/*0301

NT = not tested. a According to the Myasthenia Gravis Foundation of America (Jaretzki et al., 2000). b Antigenic peptides recognized by P3A+-stimulated T cell lines. c Determined based on blocking experiments with anti-HLA-DR and anti-HLA-DQ mAbs.

rP3A+ /rP3A ratio

Antigenic P3A peptideb

HLA class II restrictionc

1.6 1.7 1.7 1.0 1.1

p5 p5, p4, p7, p6,

NT NT HLA-DQ HLA-DQ NT

p6 p5, p6 p8 p7

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shown in the table, all five MG patients who responded to the P3A peptides had common clinical features, including thymoma and late-onset disease (age at onset after 40 years; Suzuki et al., 2001), although the combination of thymoma and late-onset MG was not found in any of the nine MG patients in whom the P3A peptide-induced T cell response was not detected. The difference in the frequency between two patient groups was statistically significant ( p = 0.0005).

4. Discussion In this study, autoreactive T cell responses to the extracellular domain of AChRa with and without the P3Aencoded region were examined using recombinant fragments and synthetic peptides in MG patients and healthy donors. A T cell proliferative response to recombinant AChRa fragments was detected in MG patients including those without anti-AChRa antibodies and healthy donors at similar frequencies. The AChRa-reactive T cells of MG patients and healthy individuals had certain features in common, such as a predominant CD4+ phenotype and a dominant restriction by HLA-DR with minor contribution by HLA-DQ, but differed in the following respects: (1) the T cell proliferative responses induced by recombinant AChRa fragments were dose-dependent in MG patients and healthy controls, but the antigen concentration required for significant response was much higher in healthy controls compared to MG patients; and (2) T cells responsive to the P3A-specific region were exclusively detected in MG patients, especially those with thymoma and late-onset disease. Because pure preparations of native AChRa subunit are not available, preparation of the antigen used to stimulate T cells in vitro poses considerable problems (Hawke et al., 1996). Although many groups have used a pool of overlapping synthetic peptides covering all or part of the AChRa sequence as a stimulating antigen, some investigators have successfully propagated specific CD4+ T cell lines by using purified recombinant AChRa fragments expressed in bacteria (Hawke et al., 1996; Conti-Fine et al., 1998; Diethelm-Okita et al., 1998). However, it has been suggested that a trace amount of bacterial components contaminating in recombinant protein preparation evokes background T cell responses especially in preprimed people (Willcox et al., 1993). We believe that AChRa-specific T cell responses were properly evaluated in this study based on the following evidences: (1) T cell proliferative response induced by MalBP, which is presumed to contain bacterial contaminants similar to rP3A+ and rP3A preparations, was minimal in the majority of MG patients and controls; and (2) T cells primed with recombinant AChRa fragment showed significant responses to synthetic peptides encoding the AChRa sequences.

The results of this study indicate that AChRa-reactive CD4+ T cells are a component of the T cell repertoire of the majority of healthy individuals. This finding is analogous to the results of previous studies that reported the presence of autoreactive T cells to recombinant fragments or synthetic peptides of AChRa in the peripheral blood of healthy individuals (Brocke et al., 1988; Diethelm-Okita et al., 1998; Matsuo et al., 1995). A possible explanation for this phenomenon is the recognition of ‘‘cryptic’’ determinants, not produced from native AChR molecules by antigen processing under normal circumstances, by AChRa-reactive T cells present in the normal T cell repertoire (Lanzavecchia, 1995). In this regard, Matsuo et al. (1995) showed that T cell lines reactive with the AChRa peptide established from healthy individuals did not recognize peptides naturally generated from whole intact AChRa by antigen processing. T cells responsive to ‘‘cryptic’’ self-determinants remain naive in vivo due to lack of antigenic stimulation under normal circumstances but would become activated and induce pathogenic autoimmune responses when the ‘‘cryptic’’ determinants were efficiently presented (Lanzavecchia, 1995). The mechanisms of antigen exposure which resulted in the activation of these T cells in vivo are still unknown, but the possibilities include factors that affect normal antigen processing, such as overexpression of the P3A+ isoform of AChRa in thymomas. However, it has been claimed that ‘‘cryptic’’ epitopes identified using T cell lines selected by stimulation with synthetic peptides, or recombinant fragments may be synthetic artifacts (Nagvekar et al., 1999). Alternatively, it is possible that thymomas generate T cell responsive to AChRa, but fail to tolerize them properly, as suggested by Nagvekar et al. (1998). An antigen dose dependency in T cell proliferative responses to recombinant AChRa fragments was different between MG patients and healthy individuals. In other words, a strong T cell response to AChRa could be induced in bulk PBMC cultures by a lower concentration of antigen in MG patients than compared to healthy individuals. This can be explained by hyperactivity of either T cells or antigen-presenting cells in MG patients. Further studies in which T cells and antigen-presenting cells isolated from a pair of HLA-matched MG and healthy individuals are mixed in different combinations are necessary to confirm this hypothesis. A number of distinct T cell epitopes have been identified in the P3A isoform of AChRa by several investigator groups (Hawke et al., 1996; Conti-Fine et al., 1998; Brocke et al., 1988; Oshima et al., 1990; Manfredi et al., 1994). However, we have demonstrated the presence of T cells specific to the P3A-encoded sequences, and these autoreactive T cells were exclusively detected in a subset of MG patients with thymoma and late-onset disease. Taken together, with the accumulating evidence of upregulated expression of the P3A+ isoform of AChRa in abnormal thymus in MG patients (Andreetta et al.,

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1997; Wakkach et al., 1996; Zheng et al., 1999; Wilisch et al., 1999), the following scenario can be proposed for the initiation of the autoimmune response to AChR in a subset of MG patients. Overexpression of the P3A+ isoform of AChRa in the abnormal thymus induces the expression of previous ‘‘cryptic’’ determinants encoded by the P3A exon, resulting in the activation of T cells responsive to the P3A-encoded sequence present in the T cell repertoire of susceptible individuals. Once the autoimmune response to the P3A-specific region is induced, the immune response may subsequently diversify to recognize other areas on the entire AChR complex, to which there was previously T cell tolerance (‘‘epitope spreading’’) (Lehmann et al., 1992; Fatenejad et al., 1993). On the other hand, Nagvekar et al. (1999) concluded that many of T cell responses to AChR peptides reported previously may likewise be neither truly autoimmune nor disease-specific. In regards to this point, it would be interesting to perform serial examination of the T cell responses to the P3A+ and P3A isoforms in newly diagnosed MG patients. Two independent T cell epitopes were identified within the P3A-encoded region: p5 (AA6+ – 20+) and p7 (AA16+ – 25+/59 –63), and both appeared to be restricted by HLADQ. The latter epitope or an equivalent region has been reported to be recognized by T cell clones generated from Caucasian patients with early-onset MG in the context of DQ5 (Harcourt et al., 1993; Matsuo et al., 1995) and by a T cell clone generated from a Japanese patient with infantonset MG in the context of DQ6 (DQB1*0604) (Kanai et al., 1997). On the other hand, AA6+ – 20+ in the middle of the P3A-encoded region has never been reported as a T cell epitope, and it is likely that this epitope is recognized in the context of DQB1*0402. This partly explains the association between the preferential response to rP3A+ and DQ4 in bulk PBMC cultures from MG patients. Moreover, DQB1*0402 has been reported to be associated with thymoma and lateonset MG in Japanese (Suzuki et al., 2001). In summary, our study demonstrated the presence of autoreactive T cells specific to the P3A-encoded sequences exclusively in MG patients with thymoma and late-onset disease. Further studies to analyze the process that primes these autoreactive T cells in MG patients in vivo will be useful in clarifying the pathogenesis of MG.

Acknowledgements We thank Y. Okazaki and K. Kimura for their excellent technical assistance, Dr. T. Fujita and Dr. K. Dan for preparing the synthetic peptides, and Dr. A. Koto, Dr. T. Amano, Dr. N. Tanahashi, Dr. J. Hamada, and Dr. S. Nogawa for coordinating the blood specimen collection. This work was supported by a grant from the Japanese Ministry of Education, Science, Sports and Culture and the Keio University Medical Science Fund.

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