Generation of acetylcholine receptor-specific human T cell lines using heterobifunctional antibody-targeted antigen presentation

Journal of Neuroimmunology 99 Ž1999. 114–121 www.elsevier.comrlocaterjneuroim

Generation of acetylcholine receptor-specific human T cell lines using heterobifunctional antibody-targeted antigen presentation Dequn Wang, Michael W. Nicolle

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Department of Clinical Neurological Sciences and Microbiology and Immunology, UniÕersity Campus, London Health Sciences Centre, UniÕersity of Western Ontario, 339 Windermere Rd, London, Ontario, Canada N6A 5A5 Received 5 October 1998; received in revised form 1 June 1999; accepted 2 June 1999

Abstract Characterizing AChR-specific T lymphocyte clones is an important step towards the ability to induce antigen-specific tolerance in myasthenia gravis ŽMG.. However, the limited supply of relatively inefficient autologous antigen presenting cells ŽAPCs. makes establishing AChR-specific T lymphocyte lines difficult. In this study we targeted AChR to autologous surface IgMq ŽsIgMq. APCs using heterobifunctional antibodies Žbi-Ab. consisting of anti-sIgM linked to anti-AChR antibodies. FACScan analysis and whole cell-based radioimmunoassay ŽRIA. showed binding of bi-AbrAChR conjugates to sIgMq APCs. Using antigen targeting, AChR-presentation to a well-characterized AChR-specific T cell clone, and to T cell lines raised de novo from MG thymocytes, was improved. Thus, antigen targeting using bi-Ab improved the efficiency of presentation of the scarce autoantigen AChR, suggesting that this method might allow the use of relatively impure antigen preparations and normally inefficient non-antigen-specific APCs, including those which can be immortalized, to accelerate the characterization of the AChR epitopes recognized by pathogenic T helper lymphocytes. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Myasthenia gravis; Acetylcholine receptor; Antigen presentation; T lymphocytes; Antigen targeting

1. Introduction Myasthenia gravis ŽMG. is a human autoimmune disease in which antibodies against skeletal muscle AChR interfere with neuromuscular transmission, resulting in fatigable muscle weakness, the clinical hallmark of MG. Histological abnormalities of the thymus are commonly found in MG, and the thymus is enriched in AChR-reactive T lymphocytes and AChR-antibody-producing B lymphocytes ŽWillcox and Vincent, 1988; Hohlfeld and Wekerle, 1994.. The current treatment of MG involves non-antigen-specific immunosuppression, which has significant adverse effects ŽVerma and Oger, 1992.. The induction of antigen-specific tolerance in AChR-specific T helper ŽTh . lymphocytes could interrupt the production of pathogenic anti-AChR autoantibodies, and is an appealing therapeutic possibility in MG ŽNicolle et al., 1994; Hawke et al., 1996.. However, the AChR is a large protein and therefore has many potential T lymphocyte epitopes ) Corresponding author. Tel.: q1-519-6633236; fax: q1-519-6633238; E-mail: [email protected]

ŽHawke et al., 1996.. The application of an antigen-specific immunotherapy, such as the induction of tolerance using soluble MHC class II-AChR peptide complexes ŽNicolle et al., 1994. requires knowledge of which epitopes are pathogenic. To date this knowledge, which is dependent on the characterization of AChR-reactive T lymphocytes in vitro, is not available. Despite considerable efforts to establish AChR-specific T lymphocyte clones in vitro, success has been very limited ŽHawke et al., 1996.. Major obstacles have included the limited supplies and inefficiencies of suitable antigen presenting cells ŽAPCs., as well as the scarcity and impurity of intact AChR for use as antigen ŽWillcox et al., 1993.. Autologous professional APCs such as macrophages and dendritic cells are in scarce supply and can not be immortalized. Although antigen-specific B cells are highly efficient APCs ŽLanzavecchia, 1990. the precursor frequency of AChR-specific B lymphocytes in peripheral blood is low ŽLink et al., 1991.. Also problematic is the choice of the antigens. Although ideal, intact human AChR is difficult to obtain and purify in sufficient amounts for use in cell culture.

0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 9 . 0 0 1 1 3 - 7

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The use of bi-Ab, in which antibodies against human sIgM are chemically cross-linked to those against mammalian AChR would allow the use of any surface IgMq cell ŽsIgM. as an ‘antigen-specific’ APC to process and present the AChR. Surface IgMq B lymphocytes would be ideal, especially as they can be immortalized while preserving their ability to process and present antigen. We pursued this by using anti-sIgMranti-AChR bi-Ab with both conventional APCs Žirradiated autologous peripheral blood mononuclear cells ŽPBMCs.. and autologous Epstein–Barr virus-transformed lymphoblastoid cell lines ŽEBV LCLs.. The latter are especially appealing since if effective in presenting AChR, they would provide an unlimited supply of ‘antigen-specific’ APCs with which to initiate AChR-specific T lymphocyte lines. Our results suggest that the use of such targeted APCs may overcome some of the previous barriers to the initiation of AChR-reactive T lymphocyte lines.

2. Materials and methods 2.1. Preparation of AChR AChR was purified from human muscle following established techniques ŽWhiting et al., 1986.. Human gastrocnemiusrsoleus muscle, obtained at limb amputation, was homogenized Ž200 gm muscle in 500 ml of 10 mM phosphate buffer ŽPB., 50 mM NaCl, 0.2 mM PMSF, 0.02% sodium azide, pH 7.4; buffer A.. After homogenization, the preparation was centrifuged Ž30,000 = g for 45 min., re-homogenized and centrifuged again. The tissue pellet was suspended in extraction buffer Ž2% Triton X-100 in buffer A. and stirred gently overnight at 48C. The resulting muscle suspension was centrifuged Ž105,000 = g for 1 h., the pellet discarded and the supernatant was used for further affinity column purification. First, Br-activated sepharose 4B Ž4.5 gm; Pharmacia. and cobratoxin Ž5 mg; Sigma. were equilibrated individually with coupling buffer Ž0.1 M NaHCO 3 , 0.5 M NaCl, pH 8.3., then combined and stirred at 48C overnight. After washing with coupling buffer, the cobratoxin-bound beads were blocked Ž0.1 M Tris–HCl pH 8.0, 2 h., and washed sequentially with sodium acetate buffer Ž0.1 M sodium acetate pH 4.0, 0.1 M Tris–HCl, 0.5 M NaCl pH 8.0. and finally extraction buffer. For affinity purification, muscle extract Ž30 ml. was mixed with cobratoxin-bound beads at 48C overnight. After loading this mixture on the column ŽFlex-column, Kontes Glass, 1.5 = 15 cm., the column was washed with buffer B Ž20 mM PB, 1 M NaCl, 0.1% Triton X-100, 0.02% sodium azide.. AChR was eluted in eluting buffer Ž1 M carbachol in buffer B.. The eluted AChR was dialyzed extensively Ž20 mM PB, pH 7.4 at 48C., washed and concentrated with a molecular weight cut-off centrifuge tube ŽCentricon 30, Amicon.. The presence of

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AChR in the extract and the eluate was confirmed by RIA, and by Western blotting using anti-AChR antibodies Ždata not shown.. 2.2. Preparation of heterobifunctional antibody conjugate Hybridoma cells secreting monoclonal mAb35 rat antitorpedo AChR antibodies ŽATCC TIB175, rat IgG., which cross react with mammalian AChR, were cultured in serum-free medium ŽHybridoma-SFM, Gibco., and the supernatant was precipitated with saturated ammonium sulfate. After centrifugation Ž1 h at 20,000 = g . the precipitant was dissolved and further purified on a Sephadex G200 column Ž2.5 = 100 cm; Pharmacia. in PB. After elution, fractions containing the highest protein concentration were grouped and concentrated ŽUltrafree-50 centrifuge tube, Millipore.. Affinity-purified goat anti-human IgM ŽGHM; polyclonal anti-Fc5m . was purchased from Jackson ImmunoResearch Laboratories ŽWest Grove, PA, USA.. Antibody cross-linking was performed as modified from Segal ŽSegal and Bast, 1995.. GHM Ž2 mg. and mAb35 Ž5 mg. were dialyzed separately against borate buffer Ž0.015 M sodium borate, 0.15 M NaCl, pH 8.5. at 48C overnight. The cross-linking agent N-succinimidyl-3Ž2-pyridyldithiol.propionate ŽSPDP, Sigma. was then added Ž4 mgrml in 100% ethanol. to mAb35 and GHM separately Ž21 ml SPDP to 0.75 ml mAb35, 8.4 ml SPDP to 2.4 ml GHM.. After incubation for 1 h at room temperature, 4 mg dithiothreitol ŽDTT, Sigma. and 4 mg cytochrome c in 0.26 ml reducing buffer Ž1 M NaCl, 1 M sodium acetate, pH 4.5. was added to the GHM which was then passed through a borate buffer-equilibrated PD-10 column ŽPharmacia.. During elution, fractions up to and including the cytochrome c-containing Žred. fraction were combined and concentrated ŽCentricon-30 filter; Amicon.. To promote the formation of heterobifunctional conjugates, the reduced GHM-SPDP was combined with non-reduced mAb35-SPDP for 4 h at room temperature. The combination was then mixed with 1 mg iodoacetamide and incubated for 48 h at 48C. The cross-linked bi-Ab antibody conjugates were separated from unbound monomeric antibodies and cytochrome c using size exclusion chromatography on a Sephadex G200 column ŽPharmacia. in borate buffer, followed by extensive dialysis and concentration with PB through an Ultrafree-50 centrifuge tube to reduce cytotoxicity. 2.3. FACScan analysis To demonstrate binding of bi-Ab or its components to the B lymphocyte surface, EBV LCL Žwhich were sIgMq as shown by preliminary FACScan analysis, not shown. were incubated with bi-Ab, or with the component monomeric antibodies in equimolar amounts, followed by the addition of the appropriate secondary detecting anti-

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bodies Žanti-goat for GHM and anti-rat for mAb35.. All steps were performed at 48C and washes in 0.1% BSA–PBS unless indicated otherwise. First, EBV LCL were incubated with primary antibodies; either bi-Ab or one of the monomeric antibody components, mAb35 or GHM Ž10–25 mgrml, 2 = 10 6 cellsrtube. for 30 min. A purified mouse isotype control antibody ŽIgG1k, Sigma. was used to assess background staining. After extensive washing, the EBV LCL cells were incubated with the appropriate secondary detecting antibodies for 30 min. Fluorescein isothiocyanate ŽFITC.-conjugated mouse anti-goat ŽJackson ImmunoResearch Laboratories, 10 mgrml. was used to detect GHM binding. For mAb35, cells were incubated with biotinylated goat anti-rat ŽJackson ImmunoResearch Laboratories, 2 mgrml., washed extensively and combined with streptavidin-phycoerythrin ŽPE, Serotech; diluted 1:10. for 30 min. For all groups, after another round of washing the binding was assessed with single colour FACScan analysis ŽBecton Dickinson..

mozygous DRb 0402q EBV LCL line was used Žkindly provided by Dr. Nick Willcox, University of Oxford.. All EBV LCL cells used were non-anti-AChR antibody secretors as verified by RIA. Unless indicated otherwise, all subsequent steps were performed at 48C, and all washes, incubations and dilutions were done in Hanks buffered saline solution ŽHBSS.. Autologous PBMC or EBV LCL cells were washed twice and incubated with bi-Ab Ž25 mgrml. for 1 h. After washing twice, the cells were incubated with purified AChR Ž25 mgrml. for 1 h, then washed and incubated in RF10 at 378C for 2 h. The AChR-targeted EBV LCL were treated with mitomycin C Ž100 mgrml, 1 h at 378C., and PBMC were irradiated Ž2500 rads., and washed. After counting, AChR-targeted APCs were added to T cells. Controls consisted of APCs treated identically, but either incubated without AChR, without bi-Ab but with AChR, or without bi-Ab or AChR. 2.6. Cell-based RIA

2.4. PM-A1 AChR-specific cell clone q

PM-A1 is a well-characterized AChR-specific CD4 T cell clone, originally initiated from the thymic cells of a seropositive MG patient with recombinant AChR a-subunit polypeptide ra 37-429 ŽOng et al., 1991; Willcox et al., 1993.. Its clonal nature has been established both by extensive epitope and MHC class II mapping, and by the demonstration of clonal TCR Vb usage in the clone and several subclones ŽWillcox et al., 1993.. The clone is specific for the core epitope of the AChR a-subunit, a149-156, and responds to stimulation by DRb 0402q APCs to any form of the AChR containing this epitope, including synthetic peptides, recombinant polypeptide AChR a-subunit or intact human AChR ŽNicolle et al., 1994, 1995.. It also responds to stimulation with autologous DRb 0408q APCs and synthetic peptides or short recombinant polypeptides, but not with longer recombinant polypeptides or intact AChR ŽNicolle et al., 1995.. 2.5. Preparation of AChR-targeted antigen-presenting cells Autologous PBMCs were obtained from heparinized venous blood following standard methodology. For some experiments, EBV LCL lines were raised from MG patients. PBMCs were incubated with 50% Žvrv. culture supernatant of the B95-8 marmoset cell line ŽATCC CRL1612.. One hour later, cyclosporin A Ž1 mgrml; a kind gift of Sandoz. was added. After culture in 10% fetal bovine serum ŽFBS.-RPMI 1640 for 4 weeks, successfully transformed EBV LCL cells were expanded and eventually cryopreserved for future use. As autologous DRb 0408q APCs are unable to effectively process or present the intact AChR to the PM-A1 clone ŽNicolle et al., 1995., a ho-

Purified AChR was radiolabelled to saturation with 3000 cpm of 125 I-a-bungarotoxin Ž a BuTx; 200 mCir mmol; Amersham.rmg AChR for 1 h at room temperature. EBV LCL cells were incubated with bi-Ab Ž50 mgrml for 30 min., washed twice and then labeled with 125 I-a BuTx-labeled purified AChR Ž0.1–100 mgrml.. Controls consisted of sham-incubated cells Žno bi-Ab. to which radio-labeled 125 I-a BuTx-AChR was added. After incubation Ž48C for 30 min. the cells were washed and counted using a gamma scintillation counter ŽLKB-Wallac CliniGamma 1272; Wallac.. 2.7. Cellular proliferation T lymphocytes Ž10 4rwell. were stimulated in triplicate with AChR-targeted APCs Ž10 5rwell. in 96-well microplates. Control groups included T cells stimulated with APCs only, APCs and bi-Ab but not AChR, or with APCs and AChR Ž25 mgrml. but without bi-Ab. For the PM-A1 cell clone the recombinant AChR a-subunit polypeptide r37-429 Ž1 mgrml; a kind gift of Drs. A. Vincent and N. Willcox, University of Oxford. was also used. At 72 h after stimulation, the cells were pulsed with 1 mCi w3 Hxthymidine ŽAmersham. for 16 h, harvested ŽCell Harvester III; Tomtec. onto filtermats ŽWallac, 1450-421. and w3 Hxthymidine incorporation measured with a liquid scintillation counter Ž1450 MicroBeta; Wallac.. 2.8. Preparation of thymocyte suspension and general cell culture Thymocytes were obtained from early onset seropositive generalized myasthenics at the time of therapeutic

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mesh Ža40. using the plunger from a sterile syringe. Single cell suspensions were prepared and used for generating AChR-specific T cell lines or as a source of APCs Žmodified from Willcox et al., 1984.. Thymocytes were cultured in a 95% airr5% CO 2 incubator in RPMI 1640 with 5% normal human serum ŽRH 5 ., 2 mM glutamine, 100 Urml penicillin and 100 mgrml streptomycin ŽGibco..

2.9. Establishment of AChR-specific T cell lines

Fig. 1. Size exclusion chromatography of chemically cross-linked heterobifunctional antibodies Žbi-Ab.. The cross-linked rat anti-torpedo Žwhich cross-reacts with mammalian. AChR and goat anti-human IgM antibodies were eluted first, followed by the monomeric component antibodies and then the cytochrome c marker as shown.

thymectomy. After rinsing in sterile HBSS, the thymus was mechanically dissociated with forceps and scissors, and then gently forced through a sterile stainless steel

Thymocytes Ž2 = 10 6 . were stimulated with AChRtargeted APCs Ž4 = 10 6 . in 24-well flat-bottom culture plate ŽFalcon 3047. in 2 ml of RH 5 . When EBV LCLs were not yet available for the initial stimulation, APCs were obtained by targeting AChR to the thymocyte suspension, which contains B lymphocytes and macrophages ŽZoller, 1991., using bi-Ab and AChR as described above. After stimulation with APCs, IL-2 Ž20–40 Urml; Genzyme. was added on days 3, 6 and sometimes 9, before restimulation on days 10–14. At the end of the first stimulation cycle, viable T cells were separated on a Ficoll Hypaque density gradient and were restimulated with AChR-targeted APCs, with IL-2 added subsequently as during the initial stimulation cycle. After four cycles of

Fig. 2. FACscan analysis of bi-Ab binding to EBV-LCL cells. The binding of the monomeric components GHM Ža., mAb35 Žb. or the cross-linked bi-Ab ŽGHM cross-linked to mAb35; c. to sIgMq EBV LCLs was assessed using the secondary antibodies anti-goat for GHM Žtop. and anti-rat for mAb35 Žbottom..

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restimulation, antigen-responsiveness was assessed in a proliferation assay. 2.10. Statistical methods For comparison of subgroups in proliferation assays, a Student’s t-test was used.

3. Results 3.1. Characterization of the cross-linked antibodies Fig. 1 shows the protein concentration profile after elution from size-exclusion chromatography of cross-linked GHM and mAb35 antibodies. The first peak, appearing before the monomeric antibody components, represents the cross-linked bi-Ab Žcf. ŽSegal and Bast, 1995.. The yield of bi-Ab was approximately 50% of the total input of component antibodies. To determine the ability of the cross-linked bi-Ab to bind human B cell sIgM, FACScan analysis was performed ŽFig. 2.. EBV LCL were incubated with GHM ŽFig. 2a., mAb35 ŽFig. 2b. or bi-Ab ŽFig. 2c. and then secondary detecting antibodies to detect the GHM ŽFig. 2 top. or mAb35 ŽFig. 2, bottom. antibody components. As expected, GHM bound to EBV either alone or when part of bi-Ab ŽFig. 2a and c, top. whereas mAb35 was not detected with this secondary antibody ŽFig. 2b top.. Similarly, GHM was not detected with the anti-rat secondary ŽFig. 2a, bottom., and mAb 35 alone did not bind to the cell surface, ŽFig. 2b, bottom.. However, the binding of mAb35 to the B lymphocyte surface was detected as part of bi-Ab when it was attached to the cell surface via GHM ŽFig. 2c, bottom.. These results confirm that the intact heterobifunctional antibody complex bound to the B lymphocyte surface. To demonstrate that mAb35, when bound to the B cell surface as part of bi-Ab, could still bind its AChR ligand, a whole cell RIA was used. As shown in Table 1, when radiolabelled AChR was targeted to EBV LCL surfaces with bi-Ab there was a dose-dependent increase in cell radioactivity above background in the absence of bi-Ab targeting.

Fig. 3. Proliferation of AChR-specific T cell clone PM-A1 after stimulation with Ža. APCs ŽU , the difference between the last two groups was significant w p- 0.05x. or Žb. after stimulation with EBV LCLs with or without bi-Ab AChR targeting. T, PM-A1 clone T cells. Px, irradiated PBMCs. AChR, enriched intact human AChR. bi-AbrAChR, AChR bound to heterobifunctional bi-Ab ŽGHM and mAb35. conjugate. EBV, DRb 0402q EBV-LCL cells; bi-Ab, heterobifunctional antibody conjugate alone.

3.2. Stimulation of an AChR-specific T clone with AChR-targeted APCs PM-A1 cells were used to determine the effectiveness of bi-Ab-targeted AChR presentation by DRb 0402q APCs. PM-A1 proliferation to stimulation with intact AChR, normally low without antigen-targeting, was im-

Table 1 Cell-based RIA of radiolabeled AChR binding to EBV-LCL cells through bi-Ab. EBV-LCL cells were pre-incubated with or without bi-Ab for 30 min at 48C before 125 I-bungarotoxin-labeled purified human AChR was added. Cells were then washed and countered by g-scintillation counting Cell RIA D cpmrmillion cells AChR Žmgrml. 125

bi-Abr I-a BuTx AChR 125 I-a BuTx AChR only Specific binding

10 2

10 1

10 0

10y1

2383 1287 1096

600 565 35

0 0 0

0 0 0

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proved by antigen targeting when irradiated PBMCs were used as APCs ŽFig. 3a.. Similarly, when using DRb 0402q EBV LCL cells as APCs, the response of PM-A1 cells to intact AChR was significantly greater when targeted with bi-Ab, as compared to stimulation in the absence of antigen-targeting which did not produce a response above background ŽFig. 3b and work not shown.. There was no response to stimulation with DRb 0408q EBV LCLs and AChR, with or without bi-Ab targeting Žnot shown.. Inexplicably, the binding of bi-Ab to EBV LCL cells increased the proliferation of the clonal AChRreactive PM-A1 cells even in the absence of AChR, although always to a lesser degree than the AChR-specific response ŽFig. 3b; see Section 4.. 3.3. Generation of AChR-reactiÕe T cell lines using bi-Ab AChR targeting Using autologous AChR-targeted APCs, AChR-specific T cell lines were generated from the thymocytes of two early onset seropositive MG patients. For the first ŽJP., after four cycles of stimulation with autologous PBMCs and bi-AbrAChR a significant proliferative response to PBMCs and bi-AbrAChR was seen ŽFig. 4. which was much greater than that to PBMCs and either bi-Ab or AChR alone. Although initiated by stimulation with PBMCs, this line responded much less well to stimulation with EBV LCLs and bi-AbrAChR Žnot shown.. Results for the second AChR-reactive line ŽKP. raised are shown in Fig. 5. This line was initiated by stimulation of thymic cells with autologous EBV LCL cells and bi-AbrAChR. Again, stimulation with bi-AbrAChRtargeted EBV LCL cells induced considerable proliferation whereas stimulation with AChR and EBV LCL cells with-

Fig. 4. AChR-specific line JP initiated from thymic cells with PBMC bi-AbrAChR targeting. Proliferation assay on day 12 after stimulation with PBMCs Ža. or EBV LCLs Žb. and antigens as shown. Px, irradiated autologous PBMCs; T, JP clone T cells. Other abbreviations are as in Fig. 3.

Fig. 5. Proliferation assay of AChR-specific line KP, initiated from thymic cells with EBV LCL bi-AbrAChR targeting. T, KP line cells. Other abbreviations are as in Figs. 3 and 4. U , The difference between the last two groups was significant Ž p- 0.05..

out bi-Ab targeting did not. As with the clone PM-A1, the binding of bi-Ab to the EBV LCL cells without AChR also stimulated a lesser degree of proliferation of KP line cells ŽFig. 5..

4. Discussion The induction of tolerance in AChR-specific T lymphocytes is an attractive plan for antigen-specific immunotherapy in MG ŽNicolle et al., 1994.. This requires knowledge of both their MHC class II and peptide restriction, information which is lacking in MG ŽWillcox et al., 1993; Nicolle et al., 1994; Hawke et al., 1996; Bond et al., 1997.. The characterization of AChR-reactive T cell clones from MG has been painfully slow ŽWillcox et al., 1993; Hawke et al., 1996; Bond et al., 1997.. Difficulties include the availability of an appropriate source of suitable antigen ŽAChR. and antigen presenting cells ŽWillcox et al., 1993; Hawke et al., 1996.. Although synthetic AChR-derived peptides are readily available, their artificially-selected end points may not correspond to those produced in vivo after the processing of intact AChR ŽMatsuo et al., 1995.. Although synthetic recombinant polypeptides cover larger segments of the AChR subunits, allowing natural processing, their synthesis is plagued by contamination with host Escherichia coli proteins ŽWillcox et al., 1993., and their production in a prokaryotic system results in the loss of conformational structure. While intact human AChR is in theory ideal for use as antigen, it is difficult to purify in sufficiently pure, plentiful and non-toxic amounts needed for tissue culture ŽSinigaglia and Gotti, 1983; Hawke et al., 1996.. Even with a suitable supply of AChR, autologous APCs are required until the line is characterized, and a suitable

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MHC class II-matched APC cell line identified. This depends on the repeated venipuncture of the original T cell donor, the logistics of which is often limiting. Conventionally APCs are obtained from irradiated PBMCs, so that only macrophages and perhaps dendritic cells continue to function as APCs. Both are scarce and cannot be immortalized. However, antigen-specific B lymphocytes are highly efficient APCs after capturing soluble antigen through their antigen-specific sIg’, and arguably might be the most relevant APC in vivo for Th lymphocyte activation in a humorally-mediated disease such as MG ŽLanzavecchia, 1985, 1990; Van Der Pouw Kraan et al., 1989; Clark and Ledbetter, 1994.. The availability of an immortal autologous cell line for use as APCs to raise AChR-reactive T lymphocyte lines would be invaluable, and EBV-transformed B lymphocytes could serve in this role. Without antigen-targeting non-antigen-specific EBV LCLs process andror present AChR poorly Žsee Section 3.. The scarcity of AChR-specific B lymphocytes Žestimated at 1r14,000 PBMCs; Link et al., 1991. lessens the chances of obtaining a stable transformed AChR-specific B lymphocyte line ŽKalsi and Isenberg, 1992.. However, any sIgMq B lymphocyte could be used as an antigen-specific APC through antigen-targeting, in which an antigen is directed to sIgM using chemically cross-linked heterobifunctional antibodies ŽSnider and Segal, 1987; Fossum et al., 1992; Segal and Bast, 1995.. Previous work suggests that antigen-targeting improves the efficiency of AChR presentation. The inclusion of autologous anti-AChR antibody-containing serum improved Th lymphocyte responses to AChR, perhaps through Fc receptor ŽFcR.-mediated uptake of AChRrantibody complexes by macrophages ŽRichman et al., 1976; ContiTronconi et al., 1979; Schalke et al., 1985.. AChR bound to paramagnetic beads with monoclonal anti-AChR antibodies improved the efficiency of antigen presentation by several fold ŽHawke et al., 1992.. The use of heterobifunctional antibodies to target AChR to chemically fixed rat splenic B lymphocytes followed by fixation allowed the induction of antigen-specific tolerance ŽReim et al., 1992., and in the absence of fixation allowed effective antigen presentation ŽReim et al., 1993.. This work is the first demonstration of more efficient antigen presentation to human MG AChR-specific T cells using AChR-targeted autologous non-antigen specific APCs. Using FACScan analysis and cell-based RIA we first demonstrated binding of heterobifunctional bi-Abr AChR to the EBV LCL surface. We then determined that antigen-targeting improved the efficiency of presentation to a previously well-characterized AChR-specific T h lymphocyte clone, as has been shown in other systems ŽSnider and Segal, 1987; Fossum et al., 1992; Reim et al., 1993.. With AChR-targeting we also initiated two other promising AChR-specific cell lines. One, raised with AChR-targeted irradiated PBMCs, did not respond to stimulation with AChR-targeted EBV LCLs ŽFig. 4., sugges-

ting that the epitopes generated from the processing of AChR by PBMC-derived APCs were not generated by EBV LCL processing. Notably, antigen-targeting also improved the efficiency of AChR presentation by macrophages ŽFigs. 3a and 4., perhaps via FcR uptake of the bi-AbrAChR complex, presumably also the mechanism of increased efficiency in the presence of autologous anti-AChR antibody-containing serum ŽConti-Tronconi et al., 1979.. The ‘background’ T lymphocyte proliferation with EBV LCLs and bi-Ab in the absence of AChR, was of practical concern in that the bi-Ab used might be antigenic, and that lines raised would react to the component antibodies and not the AChR. However, in the small number of cell lines raised to date in our laboratory with this approach the lack of response to bi-Ab alone using PBMCs as APCs was reassuring in terms of the specificity of this response. The fact that this apparent non-AChR specific stimulation was observed in the clonal AChR-reactive PM-A1 cell line suggests that this represents non-antigen specific stimulation by bi-Ab, possibly through the release of stimulatory cytokines by EBV LCLs after sIgM cross-linking. This system has several practical advantages. The use of EBV LCLs provides an unlimited supply of APCs, and the Th lymphocyte epitopes generated after B lymphocyte processing may be unique and more relevant to those generated in vivo. The specificity of the monoclonal antiAChR antibody used allows the heterobifunctional antibodies to extract AChR from relatively impure antigen preparations. The use of monoclonal antibodies against synthetic recombinant polypeptides would allow the use of these less-toxic and more readily available AChR sequences. AChR-targeting to large numbers of EBV LCLs would also provide a large supply of MHC class II molecules from which to elute AChR-derived peptides after processing, in order to characterize the relevant epitopes while circumventing the need to initiate T h lymphocyte lines in culture. Indeed, using such an approach, soluble MHC class II complexes could potentially be used to induce antigen-specific tolerance providing that the occupancy by AChR-derived peptides was sufficient ŽNicolle et al., 1994.. Thus, the technique of using heterobifunctional antibodies might overcome some of the difficulties present in attempting to initiate T lymphocyte lines against scarce autoantigens such as the AChR. In time this would allow a more accurate understanding of the range of pathogenic epitopes on the AChR which are recognized by T lymphocytes, and which would have to be targeted in an antigenspecific immunotherapy.

Acknowledgements We are grateful to Drs. Angela Vincent and Nick Willcox in the laboratory of Professor John Newsom-Davis

D. Wang, M.W. Nicoller Journal of Neuroimmunology 99 (1999) 114–121

ŽUniversity of Oxford. for providing us with the PM-A1 clone, peptide and recombinant antigen. This work was supported by a research grant from the Muscular Dystrophy Association of Canada, and by support from the Medical Research Council of Canada ŽMWN..

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