Optimization of a peptide-based protocol employing IL-7 for in vitro restimulation of human cytotoxic T lymphocyte precursors
Optimization of a peptide-based protocol employing IL-7 for in vitro restimulation of human cytotoxic T lymphocyte precursors
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DOI：
10.1016/s0022-1759(97)00177-4
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发表时间：
1997-12-15
影响因子：
2.2
通讯作者：
Rowland-Jones, S
中科院分区：
文献类型：
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作者：
Lalvani, A;Dong, T;Rowland-Jones, S
用于体外再刺激人细胞毒性T淋巴细胞（CTL）前体（CTLp）的多种不同方法正在使用中。我们的目的是提高外周血中循环人CTLp的检测。我们已经开发了一种标准化和高效的方法来重新刺激CTLp。合成肽用于再刺激来自外周血单核细胞（PBMC）的同源CTLp，产生能够赖氨酸肽脉冲的酸效应CTL和病毒感染的靶标。评价了几个参数对A型流感病毒、EBV和HIV-1特异性CTL的影响，并确定了产生CTL的最佳肽浓度。用IL-7补充初始培养物大大增强了所有测试肽的肽特异性裂解活性，并描绘了IL-7的剂量-反应关系。使用肽-MHC I类分子四聚体对携带同源T细胞受体的T细胞进行染色的新技术允许在体外再刺激期间计数抗原特异性CD 8 + CTL; IL-7补充选择性地扩增肽特异性CD 8 + CTL的群体。重要的是，该方案虽然增强了次级CTL的再刺激和抒情活性，但在体外不诱导初级CTL。在该系统中产生CTL的效率提高，显著提高了CTL培养和Cr-51释放试验检测低水平CTL活性的灵敏度。(C)1997年Elsevier Science B.V.
A variety of different methods for the in vitro restimulation of human cytotoxic T lymphocyte (CTL) precursors (CTLp) are in use. Our aim was to enhance the detection of circulating human CTLp in peripheral blood. We have developed a standardized and highly efficient method for restimulating CTLp. Synthetic peptides were used to restimulate cognate CTLp from peripheral blood mononuclear cells (PBMC), acid effector CTL capable of lysine peptide-pulsed and virus infected targets were generated. The effects of several parameters on CTL specific for influenza A, EBV and HIV-1 were evaluated, and the optimum peptide concentration for CTL generation was established. Supplementation of initial cultures with IL-7 greatly enhanced peptide-specific lyric activity for all peptides tested and the dose-response relationship for IL-7 was delineated. A novel technique using peptide-MHC class I molecule tetramers to stain T cells bearing cognate T cell receptors permitted enumeration of antigen-specific CD8 + CTL during in vitro restimulation; IL-7 supplementation selectively expanded the population of peptide-specific CD8 + CTL. Importantly, this protocol, whilst enhancing the restimulation and lyric activity of secondary CTL, does not induce primary CTL in vitro. The improved efficiency with which CTL are generated in this system substantially enhances the sensitivity of CTL culture and the Cr-51 release assay to detect low levels of CTL activity. (C) 1997 Elsevier Science B.V.