RESPONSE OF CD4(-CELLS FROM MYASTHENIC PATIENTS AND HEALTHY-SUBJECTS OF BIOSYNTHETIC AND SYNTHETIC SEQUENCES OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR() T)

We investigated the suitability of pools of overlapping synthetic pept ides spanning the complete alpha(1) subunit sequence of the human musc le acetylcholine receptor (AChR) (alpha(1) pool) or the extracellular domain (residues 1-218, alpha(1)1-218 pool), and of biosynthetic alpha 1 constructs from E. coli, as stimulants of human CD4(+) cells from m yasthenia gravis (MG) patients and healthy subjects. A construct corre sponding to residues alpha(1)1-209 was obtained as solubilized inclusi on bodies (ib alpha(1)1-209), or purified by SDS gel electrophoresis ( pur alpha(1),1-209). A second construct included the extracellular, cy toplasmic and carboxyl-terminal domains plus histidine residues, and w as obtained as inclusion bodies (ib alpha(1)NoTrans) or purified by ge l permeation and histidine tag affinity chromatography (pur alpha(1)No Trans). A biosynthetic extracellular domain of the neuronal AChR alpha (7) subunit (ib alpha(7)1-206) isolated from E. coli as inclusion bodi es served as control for bacterial contaminants. We used ib alpha(1)1- 209, pur alpha(1)1-209 and peptide pools to propagate CD4(+) lines fro m two MG patients. The lines obtained using pur alpha(1)1-209 and the peptide pools recognized the peptide pools and alpha(1) constructs tes ted well, but ib alpha(7)1-206 poorly or not at all. These lines recog nized peptides known to form CD4(+) epitopes in these patients. The ib alpha(1)1-209 lines recognized ib alpha(1)1-209 and ib alpha(7)1-206 strongly, but recognized poorly pur alpha(1)1-209 and the alpha(1)1-21 8 pool. We propagated T-cell lines from a healthy subject using pur al pha(1)1-209 and ib alpha(1)1-209. The pur alpha(1)1-209 line recognize d pur alpha(1)1-209 and the alpha(1)1-218 pool, but not ib alpha(1)1-2 09 or ib alpha(7)1-206. The ib alpha(1)1-209 line recognized ib alpha( 1)1-209 and ib alpha(7)1-206, but not pur alpha(1)1-209 or the alpha(1 )1-218 pool. We tested blood CD4(+) cells from six MG patients and eig ht healthy subjects with ib alpha(1)1-209, pur alpha(1)1-209, the alph a(1)1-218 pool and-in the healthy subjects-also ib alpha(7)1-206, ib a lpha(1)NoTrans and pur alpha(1)NoTrans. In both populations, the alpha (1)1-218 pool elicited low and sporadic responses, while the construct s elicited clear responses that were frequently higher for ib alpha(1) 1-209 than pur alpha(1)1-209. The responses to ib alpha(7)1-206 were s trong and comparable to those to ib alpha(1)1-209, ib alpha(1)NoTrans, and pur alpha(1)NoTrans. These results indicate that even purified co nstructs from E. coli contain bacterial contaminants recognized by CD4 (+) cells. They should not be used to test unselected blood CD4(+) cel ls, because they may evoke strong CD4(+) responses to the bacterial an tigens. Purified recombinant sequences may be suitable for propagation of CD4(+) cell lines, if the specificity of the Lines can be verified using different antigen prepar; ations. Short synthetic peptide seque nces can be safely used for propagation of specific CD4(+) cells. Alth ough they are poor stimulants for unselected blood CD4(+) cells, the l ow responses they elicit are probably due to these cells. (C) 1998 Aca demic Press Limited.