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.
Bibliographical noteFunding Information:
Supported by the NINDS grant NS 23919 and a research grant from the Muscular Dystrophy Association (to B.M.C.-F.); by the NIH grant NS 11323 and grants from the Muscular Dystrophy Association and the Smokeless Tobacco Research Council, Inc. (to J.M.L.); and by the NIH grant NS 01903 (to G.B.W.).
- Bacterial expressed antigens
- Myasthenia gravis
- Nicotinic receptor
- Synthetic T- epitopes
- T-cell epitopes