title: Response of CD4+ T cells from myasthenic patients and healthy subjects of biosynthetic and synthetic sequences of the nicotinic acetylcholine receptor.
abstract: We investigated the suitability of pools of overlapping synthetic peptides spanning the complete alpha 1 subunit sequence of the human muscle acetylcholine receptor (AChR) (alpha 1 pool) or the extracellular domain (residues 1-218, alpha 11-218 pool), and of biosynthetic alpha 1 constructs from E. coli, as stimulants of human CD4+ cells from myasthenia gravis (MG) patients and healthy subjects. A construct corresponding to residues alpha 11-209 was obtained as solubilized inclusion bodies (ib alpha 11-209), or purified by SDS gel electrophoresis (pur alpha 11-209). A second construct included the extracellular, cytoplasmic and carboxylterminal domains plus histidine residues, and was obtained as inclusion bodies (ib alpha 1NoTrans) or purified by gel permeation and histidine tag affinity chromatography (pur alpha 1NoTrans). A biosynthetic extracellular domain of the neuronal AChR alpha 7 subunit (ib alpha 71-206) isolated from E. coli as inclusion bodies served as control for bacterial contaminants. We used ib alpha 11-209, pur alpha 11-209 and peptide pools to propagate CD4+ lines from two MG patients. The lines obtained using pur alpha 11-209 and the peptide pools recognized the peptide pools and alpha 1 constructs tested well, but ib alpha 71-206 poorly or not at all. These lines recognized peptides known to form CD4+ epitopes in these patients. The ib alpha 11-209 lines recognized ib alpha 11-209 and ib alpha 71-206 strongly, but recognized poorly pur alpha 11-209 and the alpha 11-218 pool. We propagated T-cell lines from a healthy subject using pur alpha 11-209 and ib alpha 11-209. The pur alpha 11-209 line recognized pur alpha 11-209 and the alpha 11-218 pool, but not ib alpha 11-209 or ib alpha 71-206. The ib alpha 11-209 line recognized ib alpha 11-209 and ib alpha 71-206, but not pur alpha 11-209 or the alpha 11-218 pool. We tested blood CD4+ cells from six MG patients and eight healthy subjects with ib alpha 11-209, pur alpha 11-209, the alpha 11-218 pool and--in the healthy subjects--also ib alpha 71-206, ib alpha 1NoTrans and pur alpha 1NoTrans. In both populations, the alpha 11-218 pool elicited low and sporadic responses, while the constructs elicited clear responses that were frequently higher for ib alpha 11-209 than pur alpha 11-209. The responses to ib alpha 71-206 were strong and comparable to those to ib alpha 11-209, ib alpha 1NoTrans, and pur alpha 1NoTrans. These results indicate that even purified constructs from E. coli contain bacterial contaminants recognized by CD4+ cells. They should not be used to test unselected blood CD4+ cells, because they may evoke strong CD4+ responses to the bacterial antigens. Purified recombinant sequences may be suitable for propagation of CD4+ cell lines, if the specificity of the lines can be verified using different antigen preparations. Short synthetic peptide sequences can be safely used for propagation of specific CD4+ cells. Although they are poor stimulants for unselected blood CD4+ cells, the low responses they elicit are probably due to these cells.
authors.affiliation: Department of Biochemistry, College of Biological Sciences University of Minnesota, St Paul 55108, USA.
authors.forename: B
authors.initials: B
authors.lastname: Diethelm-Okita
authors.forename: G B
authors.initials: GB
authors.lastname: Wells
authors.forename: A
authors.initials: A
authors.lastname: Kuryatov
authors.forename: D
authors.initials: D
authors.lastname: Okita
authors.forename: J
authors.initials: J
authors.lastname: Howard
authors.forename: J M
authors.initials: JM
authors.lastname: Lindstrom
authors.forename: B M
authors.initials: BM
authors.lastname: Conti-Fine
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000276:immunology; D016477:Artifacts; D001327:Autoimmune Diseases / Q000276:immunology* / Q000473:pathology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000473:pathology; D002478:Cells, Cultured; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D006801:Humans; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000276:immunology; D009157:Myasthenia Gravis / Q000276:immunology* / Q000473:pathology; D010446:Peptide Fragments / Q000096:biosynthesis / Q000138:chemical synthesis / Q000276:immunology*; D011978:Receptors, Nicotinic / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology
chemical_list: D000942:Antigens, Bacterial; D000939:Epitopes; D009124:Muscle Proteins; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; D011993:Recombinant Fusion Proteins