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 a1 subunit sequence of the human muscle acetylcholine receptor (AChR) (a1 pool) or the extracellular domain (residues 1-218, a1-218 pool), and of biosynthetic a1 constructs from E. coli, as stimulants of human CD4+ cells from myasthenia gravis (MG) patients and healthy subjects. A construct corresponding to residues a1-209 was obtained as solubilized inclusion bodies (iba11-209), or purified by SDS gel electrophoresis (pura1-1209). A second construct included the extracellular, cytoplasmic and carboxylterminal domains plus histidine residues, and was obtained as inclusion bodies (iba1NoTrans) or purified by gel permeation and histidine tag affinity chromatography (pura1NoTrans). A biosynthetic extracellular domain of the neuronal AChR a7 subunit (iba71-206) isolated from E. coli as inclusion bodies served as control for bacterial contaminants. We used iba11-209, pura1-209 and peptide pools to propagate CD4+ lines from two MG patients. The lines obtained using pura1-209 and the peptide pools recognized the peptide pools and a1 constructs tested well, but iba71-206 poorly or not at all. These lines recognized peptides known to form CD4+ epitopes in these patients. The iba1-209 lines recognized iba1- 209 and iba71-206 strongly, but recognized poorly pura11-209 and the a1- 218 pool. We propagated T-cell lines from a healthy subject using pura1-209 and iba11-209. The pura11-209 line recognized pura1-209 and the a11-218 pool, but not iba11-209 or iba71-206. The iba11-209 line recognized iba11-209 and iba71-206, but not pura11-209 or the a11-218 pool. We tested blood CD4+ cells from six MG patients and eight healthy subjects with iba11-209, pura11-209, the a11-218 pool and in the healthy subjects also iba71-206, iba1NoTrans and pura1NoTrans. In both populations, the a11- 218 pool elicited low and sporadic responses, while the constructs elicited clear responses that were frequently higher for iba11-209 than pura11-209. The responses to iba71-206 were strong and comparable to those to iba11- 209, iba1NoTrans, and pura1NoTrans. 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.