The ELISPOT assay is increasingly being used for the monitoring of the induction of antigen-reactive T cells in cancer vaccination trials. In order to evaluate the reliability of T cell frequency analysis with the ELISPOT assay, a comparative study was performed in four European laboratories. Six samples from healthy subjects were analyzed for the frequency of influenza-reactive CD8+ T cells in peripheral blood mononuclear cells (PBMC) by IFNgamma-ELISPOT assay. In addition, one laboratory determined cytotoxic T cell precursor (CTL) frequencies in these samples by limiting dilution chromium-release assay (LDA), and three laboratories performed a variant of the LDA, the multiple microculture assay (MMA). Consistent frequencies of influenza peptide-reactive T cells were obtained with the ELISPOT assay in all four laboratories. The numbers detected by ELISPOT assay correlated closely with those determined by LDA. In contrast, the frequencies obtained with the MMA differed considerably and showed little correlation with the other two assays. This study shows that it is possible to use the ELISPOT assay to determine with reliability antigen-reactive T cells in a multicenter setting. We suggest that this assay may be suitable for monitoring cancer vaccine trials.

译文

ELISPOT测定法越来越多地用于监测癌症疫苗接种试验中抗原反应性T细胞的诱导。为了评估使用ELISPOT测定法进行T细胞频率分析的可靠性,在四个欧洲实验室进行了比较研究。通过IFNgamma-ELISPOT测定法分析了来自健康受试者的六个样本的外周血单核细胞 (PBMC) 中流感反应性CD8 T细胞的频率。此外,一个实验室通过限制稀释铬释放测定 (LDA) 确定了这些样品中的细胞毒性T细胞前体 (CTL) 频率,三个实验室进行了LDA的变体,即多重微培养测定 (MMA)。在所有四个实验室中,通过ELISPOT测定法获得了一致的流感肽反应性T细胞频率。ELISPOT测定法检测到的数字与LDA测定的数字密切相关。相反,用MMA获得的频率差异很大,并且与其他两种测定几乎没有相关性。这项研究表明,可以使用ELISPOT测定法在多中心环境中可靠地确定抗原反应性T细胞。我们建议该测定法可能适用于监测癌症疫苗试验。

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