Dextramers:: New generation of fluorescent MHC class I/peptide multimers for visualization of antigen-specific CD8+ T cells
Dextramers:: New generation of fluorescent MHC class I/peptide multimers for visualization of antigen-specific CD8+ T cells
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DOI：
10.1016/j.jim.2006.01.006
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发表时间：
2006-03-20
影响因子：
2.2
通讯作者：
Romero, P
中科院分区：
文献类型：
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作者：
Batard, P;Peterson, DA;Romero, P
由于基于与单生物素化的I类MHC-肽单体复合物相关的抗生物素蛋白-荧光染料缀合物的“四聚体”的开发，抗原特异性T细胞的直接鉴定以及分离成为可能。原则上，一系列不同的I类MHC-肽四聚体，每个用不同的荧光染料标记，将允许同时计数尽可能多的独特的抗原特异性CD 8（+）T细胞。然而，实际上，只有藻红蛋白和别藻蓝蛋白共轭四聚体已普遍可用，多重标记施加严重的限制。为了克服这种限制，我们开发了葡聚糖多聚体，其是基于带有多个荧光素和链霉亲和素部分的葡聚糖主链的多聚体。在这里，我们展示了这些新探针在具有四种独立抗原特异性的人CD 8（+）T细胞克隆上的功能和优化。它们的应用程序相对较低的频率在外周血中的抗原特异性T细胞的分析，以及它们在荧光显微镜的使用，被证明。数据显示，右旋糖酐产生比其荧光化四聚体对应物更强的信号。因此，这些可能成为抗原特异性T细胞标记从基础研究过渡到临床应用的首选试剂。(c)2006 Elsevier B. V.保留所有权利。
Direct identification as well as isolation of antigen-specific T cells became possible since the development of '' tetramers '' based on avidin-fluorochrome conjugates associated with mono-biotinylated class I MHC-peptide monomeric complexes. In principle, a series of distinct class I MHC-peptide tetramers, each labelled with a different fluorochrome, would allow to simultaneously enumerate as many unique antigen-specific CD8(+) T cells. Practically, however, only phycoerythrin and allophycocyanin conjugated tetramers have been generally available, imposing serious constraints for multiple labeling. To overcome this limitation, we have developed dextramers which are multimers based on a dextran backbone bearing multiple fluorescein and streptavidin moieties. Here we demonstrate the functionality and optimization of these new probes on human CD8(+) T cell clones with four independent antigen specificities. Their applications to the analysis of relatively low frequency antigen-specific T cells in peripheral blood, as well as their use in fluorescence microscopy, are demonstrated. The data show that dextramers produce a stronger signal than their fluoresceinated tetramer counterparts. Thus, these could become the reagents of choice as the antigen-specific T cell labeling transitions from basic research to clinical application. (c) 2006 Elsevier B.V. All rights reserved.