Application of the ELISPOT assay to the characterization of CD8(+) responses to Epstein-Barr virus antigens.
Application of the ELISPOT assay to the characterization of CD8(+) responses to Epstein-Barr virus antigens.
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DOI：
10.1182/blood.v95.1.241
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发表时间：
2000
期刊：
影响因子：
20.3
通讯作者：
Jie Yang;Victor M. Lemas;Ian W. Flinn;Chris Krone;R. Ambinder
中科院分区：
文献类型：
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作者：
Jie Yang;Victor M. Lemas;Ian W. Flinn;Chris Krone;R. Ambinder
CD8（+）细胞在控制爱泼斯坦 - 巴尔病毒（EBV）感染中具有重要作用。我们将干扰素ELISPOT分析调整为EBV特异性CD8（+）细胞的定量分析。使用健康供体的外周血单核细胞（PBMC），我们使用EBV转换的淋巴细胞细胞系（LCLS）作为刺激剂来测量对病毒的骨料反应，作为刺激剂，以及对2个A2 A2 A2 A2限制性肽的特定反应：亚限制性肽的增长：膜蛋白-2（LMP2）肽Clgglltmv和早期裂解BMLF1肽GLCTLVAML。在所有EBV播阳供体（范围954至37 830斑点/10（6）CD8（+）细胞）中检测到LCL反应性CD8（+）细胞。在11个健康的血清阳性A2供体中，有10个（范围11至83个斑点/10（6）PBMC）中检测到LMP2肽反应性CD8（+）细胞。在检查的所有血清阳性A2供体中检测到BMLF1肽反应CD8（+）细胞（范围13至943个斑点/10（6）PBMC）。还研究了通过每周刺激LCL为治疗目的产生的细胞毒性T淋巴细胞（CTL）线。相对于PBMC，这些CTL线显示LCL反应和LMP2肽反应性CD8（+）细胞的水平显着增加，而BMLF1肽反应性CD8（+）细胞的扩展程度较小。最后，我们应用了ELISPOT测定法以监视EBV CTL线的收养。在2名患者中，输注后可以检测到LCL响应性CD8（+）细胞的短暂增加。因此，ELISPOT分析可以应用于PBMC中对EBV抗原的CD8（+）反应的分析，在离体扩展的CTL系中以及来自经体过扩展CTL系的患者的PBMC中。 （血液2000; 95：241-248）
CD8(+) cells have an important role in controlling Epstein-Barr virus (EBV) infection. We adapted the interferon-gamma ELISPOT assay to the quantitative analysis of EBV-specific CD8(+) cells. Using peripheral blood mononuclear cells (PBMCs) from healthy donors, we measured both the aggregate response to the virus, using EBV-transformed lymphoblastoid cell lines (LCLs) as stimulators, and the specific responses to 2 A2-restricted peptide epitopes: the subdominant latency membrane protein-2 (LMP2) peptide CLGGLLTMV and the early lytic BMLF1 peptide GLCTLVAML. LCL-responsive CD8(+) cells were detected in all EBV-seropositive donors (range 954 to 37 830 spots/10(6) CD8(+) cells). LMP2 peptide-responsive CD8(+) cells were detected in 10 of 11 healthy seropositive A2 donors (range 11 to 83 spots/10(6) PBMC). BMLF1 peptide-responsive CD8(+) cells were detected in all seropositive A2 donors examined (range 13 to 943 spots/10(6) PBMC). Cytotoxic T-lymphocyte (CTL) lines generated with weekly stimulation of LCLs for therapeutic purposes were also studied. Relative to PBMCs, these CTL lines showed a marked increase in the level of LCL-responsive and LMP2 peptide-responsive CD8(+) cells and a lesser degree of expansion of BMLF1 peptide-responsive CD8(+) cells. Finally, we applied the ELISPOT assay to monitor adoptive infusion of EBV CTL lines. In 2 patients examined, a transient increase in LCL-responsive CD8(+) cells could be detected after infusion. Thus, the ELISPOT assay can be applied to the analysis of CD8(+) responses to EBV antigens in PBMCs, in ex vivo expanded CTL lines, and in PBMCs from patients treated with ex vivo expanded CTL lines. (Blood. 2000;95:241-248)