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AChR-specific T cell responses to native human AChR
Euro-Myasthenia
III
TO FVE HUMAN AChR. SHB Hawke, HNA Harcourt, Vincent and J Newsom-Davis. Neurosciences Group, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.
2.~2~~~~~ECI~COT,~ELLGRESPONSES
r
Responses to native human acetylcholine receptor (AChR) by human T cell lines and Clones raised against recombinant human AChR a subunit would help to confirm their potential relevance to the pathogenesis of MG. PM-A is a DR4iDw14.2 restricted T cell line derived from myasthenic thymus using recombinant human AChR a subunit which has been mapped to ~144-156 of the human AChR a subunit sequence (see abstract - Ong et
al.
).
AChR was purified from human calf muscle by affinity chromatography with yields of 5-47 pmoles AChR per 100 mls of crude muscle extract. After dialysis we used the purified AChR to stimulate line PM-A in 'HThvmidine incorporation assays and noted si(gnificant responses- (20 -
50% of maximum responses) with very low AChR concentrations (30"1to lo-= M). In contrast higher concentrations of full length recombinant a subunit (lo-'to lo-" M) or the epitope peptide ~144-156 (10." to lo-' M) were required to achieve comparable PM-A stimulation.
2.13 BINDING OF ACETYLCHOLINE
RECEPTOR ALPHA SUBUNIT PEPTIDES TO MHC CLASS
I MOLECULES. E._&ggfn’, J. Elvin', A. Townsend',R. Mantegazza', A. Vincent", J.Newsom-Davis". ' Neurosciences Group, Inst.Mol. Medicine,John Radcliffe Hospital, Oxford (UK). * Molecular Immunology Group, Inst. Mol. Medicine, John Radcliffe Hospital, Oxford (UK). ’ Dept. Neuromuscular Disease, National Neurological Inst. “CBesta”, Milan (I). The Acetylcholine Receptor (AChR) is the target of the autoimmune response in Myasthenia Gravis (MG). Both AChR specific autoantibodies and class II restricted CD4+ T lymphocytes can be. detected in MG patients and reflect the altered equilibrium of tolerance against self-proteins. The Human Leucocyte Antigens (HLA) haplotypes Al, B8, DR3 and A3, B7, DR2 are found with increased frequency in MG patients, suggesting that both MHC class I and class II molecules could be involved in disease susceptibility. It has been shown that MHC class I-specific peptides can support the assembly of class I molecules in vitro, stabilizing a conformational change in heavy chain and its association with &-microglobulin (1). By immunopecipitation experiments with HLA-A2 molecules from the mutant human cell line, .174A2, it is possible to detect the peptides able to stabilize the class I molecule-B,m complex in a form recognized by conformational dependent mAb (2). Using this system, we have evaluated the binding of AChR synthetic peptides to HLA-A2. So far two peptides, ~310-327 and p391-417, corresponding to sequences within the cytoplasmic region of alpha AChR subunit behveen M3 and M4 transmembrane domains, have been shown to bind HLA-A2. These and other identified peptides will be used to raise AChR specific, class I restricted, CD8+ T lymphocytes from A;?-matched PBL depleted of CD4+ T cells. The functions of these cells and their possible role within immune system interaction will be evaluated using AGhR transfected A2+ EBV-LCL and A2 transfected TEi671 (AChR+) c&l% (1) A. Townsend
et al., Cell 62:285-295, 1990. (2) V. Cerundolo et al., Nature 3451449-452, 1990. xiv
III
TO FVE HUMAN AChR. SHB Hawke, HNA Harcourt, Vincent and J Newsom-Davis. Neurosciences Group, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.
2.~2~~~~~ECI~COT,~ELLGRESPONSES
r
Responses to native human acetylcholine receptor (AChR) by human T cell lines and Clones raised against recombinant human AChR a subunit would help to confirm their potential relevance to the pathogenesis of MG. PM-A is a DR4iDw14.2 restricted T cell line derived from myasthenic thymus using recombinant human AChR a subunit which has been mapped to ~144-156 of the human AChR a subunit sequence (see abstract - Ong et
al.
).
AChR was purified from human calf muscle by affinity chromatography with yields of 5-47 pmoles AChR per 100 mls of crude muscle extract. After dialysis we used the purified AChR to stimulate line PM-A in 'HThvmidine incorporation assays and noted si(gnificant responses- (20 -
50% of maximum responses) with very low AChR concentrations (30"1to lo-= M). In contrast higher concentrations of full length recombinant a subunit (lo-'to lo-" M) or the epitope peptide ~144-156 (10." to lo-' M) were required to achieve comparable PM-A stimulation.
2.13 BINDING OF ACETYLCHOLINE
RECEPTOR ALPHA SUBUNIT PEPTIDES TO MHC CLASS
I MOLECULES. E._&ggfn’, J. Elvin', A. Townsend',R. Mantegazza', A. Vincent", J.Newsom-Davis". ' Neurosciences Group, Inst.Mol. Medicine,John Radcliffe Hospital, Oxford (UK). * Molecular Immunology Group, Inst. Mol. Medicine, John Radcliffe Hospital, Oxford (UK). ’ Dept. Neuromuscular Disease, National Neurological Inst. “CBesta”, Milan (I). The Acetylcholine Receptor (AChR) is the target of the autoimmune response in Myasthenia Gravis (MG). Both AChR specific autoantibodies and class II restricted CD4+ T lymphocytes can be. detected in MG patients and reflect the altered equilibrium of tolerance against self-proteins. The Human Leucocyte Antigens (HLA) haplotypes Al, B8, DR3 and A3, B7, DR2 are found with increased frequency in MG patients, suggesting that both MHC class I and class II molecules could be involved in disease susceptibility. It has been shown that MHC class I-specific peptides can support the assembly of class I molecules in vitro, stabilizing a conformational change in heavy chain and its association with &-microglobulin (1). By immunopecipitation experiments with HLA-A2 molecules from the mutant human cell line, .174A2, it is possible to detect the peptides able to stabilize the class I molecule-B,m complex in a form recognized by conformational dependent mAb (2). Using this system, we have evaluated the binding of AChR synthetic peptides to HLA-A2. So far two peptides, ~310-327 and p391-417, corresponding to sequences within the cytoplasmic region of alpha AChR subunit behveen M3 and M4 transmembrane domains, have been shown to bind HLA-A2. These and other identified peptides will be used to raise AChR specific, class I restricted, CD8+ T lymphocytes from A;?-matched PBL depleted of CD4+ T cells. The functions of these cells and their possible role within immune system interaction will be evaluated using AGhR transfected A2+ EBV-LCL and A2 transfected TEi671 (AChR+) c&l% (1) A. Townsend
et al., Cell 62:285-295, 1990. (2) V. Cerundolo et al., Nature 3451449-452, 1990. xiv