Response of CD4+ T cells from myasthenic patients and healthy subjects of biosynthetic and synthetic sequences of the nicotinic acetylcholine receptor.
Response of CD4+ T cells from myasthenic patients and healthy subjects of biosynthetic and synthetic sequences of the nicotinic acetylcholine receptor.
复制标题
肌无力患者和健康受试者的 CD4 T 细胞对烟碱乙酰胆碱受体生物合成和合成序列的反应。
DOI：
10.1006/jaut.1998.0190
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发表时间：
1998
期刊：
影响因子：
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通讯作者：
Conti-Fine,BM
中科院分区：
文献类型：
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作者：
Diethelm-Okita,B;Wells,GB;Kuryatov,A;Okita,D;Howard,J;Lindstrom,JM;Conti-Fine,BM
我们研究了跨越人类肌肉乙酰胆碱受体（AChR）完整α 1亚基序列（α 1库）或细胞外结构域（残基1-218，α11-218库）的重叠合成肽库以及生物合成α1的适用性构建体来自大肠杆菌。大肠杆菌，作为来自重症肌无力（MG）患者和健康受试者的人CD 4+细胞的刺激物。以可溶性包涵体（ibα11 - 209）形式获得对应于残基α11 - 209的构建体，或通过SDS凝胶电泳（purα11-209）纯化。第二个构建体包括细胞外、细胞质和羧基末端结构域加上组氨酸残基，并以包涵体（ibα 1 NoTrans）形式获得或通过凝胶渗透和组氨酸标签亲和色谱法（purα 1 NoTrans）纯化。弗罗姆中分离到神经元AChR α 7亚基（ibα71-206）的一个生物合成胞外区。大肠杆菌包涵体用作细菌污染物的对照。我们使用dib α11-209、purα11-209和肽池从两名MG患者中扩增CD 4+细胞系。使用pur α11-209和肽池获得的细胞系识别肽池和α 1构建体，但识别ibα71-206较差或根本不识别。这些细胞系识别已知在这些患者中形成CD 4+表位的肽。它们对ibα 11 - 209和ib α71-206有较强的识别能力，但对purα11-209和α11-218库的识别能力较差。我们使用purα11-209和b α11-209从健康受试者中增殖T细胞系。purα11-209线可识别pur α11-209和α11-218库，但不能识别ibα11-209 oribα71-206。ibα11-209系可识别ib α11-209和ibα71-206，但不能识别纯α11-209或α11-218库。我们检测了6名MG患者和8名健康受试者的血液CD 4+细胞，其中包括ib α11-209、purα11-209、α11-218样本池，以及健康受试者中的ibα71-206、ibα 1 NoTrans和purα 1 NoTrans。在两个人群中，α11-218合并液引起的应答较低且零星，而构建体引起的明确应答通常高于pur α 11 - 209。对ib α71-206的反应较强，与ibα11-209、ibα 1 NoTrans和purα 1 NoTrans相当。这些结果表明，即使弗罗姆.大肠杆菌含有被CD 4+细胞识别的细菌污染物。它们不应用于测试未选择的血液CD 4+细胞，因为它们可能会引起对细菌抗原的强烈CD 4+反应。纯化的重组序列可能适用于CD 4+细胞系的增殖，前提是可以使用不同的抗原纯化来验证细胞系的特异性。短的合成肽序列可以安全地用于特异性CD 4+细胞的增殖。虽然它们对外周血CD 4+细胞的刺激作用很差，但它们引起的低反应可能是由于这些细胞。
We investigated the suitability of pools of overlapping synthetic peptides spanning the complete α1subunit sequence of the human muscle acetylcholine receptor (AChR) (α1pool) or the extracellular domain (residues 1-218, α11-218 pool), and of biosynthetic α1 constructs from E. coli, as stimulants of human CD4+cells from myasthenia gravis (MG) patients and healthy subjects. A construct corresponding to residues α11-209 was obtained as solubilized inclusion bodies (ibα11-209), or purified by SDS gel electrophoresis (purα11-209). A second construct included the extracellular, cytoplasmic and carboxylterminal domains plus histidine residues, and was obtained as inclusion bodies (ibα1NoTrans) or purified by gel permeation and histidine tag affinity chromatography (purα1NoTrans). A biosynthetic extracellular domain of the neuronal AChR α7subunit (ibα71-206) isolated fromE. colias inclusion bodies served as control for bacterial contaminants. We usedibα11-209, purα11-209 and peptide pools to propagate CD4+lines from two MG patients. The lines obtained usingpurα11-209 and the peptide pools recognized the peptide pools and α1constructs tested well, but ibα71-206 poorly or not at all. These lines recognized peptides known to form CD4+epitopes in these patients. Theibα11-209 lines recognized ibα11-209 andibα71-206 strongly, but recognized poorly purα11-209 and the α11-218 pool. We propagated T-cell lines from a healthy subject using purα11-209 andibα11-209. The purα11-209 line recognizedpurα11-209 and the α11-218 pool, but not ibα11-209 oribα71-206. The ibα11-209 line recognizedibα11-209 and ibα71-206, but notpurα11-209 or the α11-218 pool. We tested blood CD4+cells from six MG patients and eight healthy subjects withibα11-209, purα11-209, the α11-218 pool and—in the healthy subjects—also ibα71-206,ibα1NoTrans and purα1NoTrans. In both populations, the α11-218 pool elicited low and sporadic responses, while the constructs elicited clear responses that were frequently higher foribα11-209 than purα11-209. The responses toibα71-206 were strong and comparable to those to ibα11-209,ibα1NoTrans, and purα1NoTrans. These results indicate that even purified constructs fromE. colicontain 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 prepar-ations. 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.