Desombere I, Meuleman P, Rigole H, Willems A, Irsch J, Leroux-Roels G
Center for Vaccinology, Department Clinical Biology, Microbiology and Immunology, Ghent University and Hospital, De Pintelaan 185, 9000 Ghent, Belgium. isabelle@
J Immunol Methods. 2004 Mar;286(1-2):167-85. doi: 10.1016/j.jim.2004.01.001.
Different single-cell analyses for the detection of antigen-specific T cells based on antigen-triggered induction of cytokine production (elispot, intracellular cytokine staining, cytokine secretion assay, etc.) have been analyzed. In this paper we present the data of a thorough validation of the IFNgamma Secretion Assay (ISA, Miltenyi Biotec, Bergisch Gladbach, Germany). In this assay the secreted IFNgamma is bound to the cell surface and is then stained as an artificial surface molecule and analyzed by flow-cytometry. The introduction of five quality criteria markedly improved the reproducibility of this assay and made it very reliable (intra-assay variability<5%; inter-assay variability<20%). Recovery experiments further demonstrated that almost 100% of IFNgamma(+) labeled cells could be detected by this technology. In order to analyze which cell subsets contribute to IFNgamma-production, we compared the results obtained in different individuals after VZAg-stimulation. Three different IFNgamma-secretion patterns could be discerned. In Pattern 1 there is a predominant and almost equal contribution of T cells and NK cells with a minor contribution of CD3(+)CD56(+) and B cells. Pattern 2, which is most abundant, is characterized by a predominance of NK cells (60-70%). Pattern 3 differs from the previous one in its minor contribution of NK cells. Here T cells predominate the IFNgamma secretion. These results clearly demonstrate that the IFNgamma(+) subset distribution after VZAg-stimulation is not uniform and differs individually. Furthermore, the ISA-technology proves to be very useful in vaccine research. This was demonstrated by testing the IFNgamma(+) secretion pattern after HBsAg-stimulation in PBMC from HBsAg-vaccinated individuals.
已对基于抗原触发的细胞因子产生诱导作用来检测抗原特异性T细胞的不同单细胞分析方法（酶联免疫斑点法、细胞内细胞因子染色、细胞因子分泌测定等）进行了分析。在本文中，我们展示了对IFNγ分泌测定法（ISA，德国米尔滕yi生物技术公司，伯格isch格拉德巴赫）进行全面验证的数据。在该测定法中，分泌的IFNγ与细胞表面结合，然后作为人工表面分子进行染色，并通过流式细胞术进行分析。引入五个质量标准显著提高了该测定法的重现性，并使其非常可靠（批内变异<5%；批间变异<20%）。回收率实验进一步证明，该技术可检测到几乎100%的IFNγ(+)标记细胞。为了分析哪些细胞亚群对IFNγ的产生有贡献，我们比较了VZAg刺激后不同个体获得的结果。可以识别出三种不同的IFNγ分泌模式。在模式1中，T细胞和NK细胞有主要且几乎相等的贡献，CD3(+)CD56(+)和B细胞的贡献较小。模式2最为常见，其特征是NK细胞占主导（60 - 70%）。模式3与前一种模式的不同之处在于NK细胞的贡献较小。在这里，T细胞在IFNγ分泌中占主导。这些结果清楚地表明，VZAg刺激后IFNγ(+)亚群分布并不均匀，且个体之间存在差异。此外，ISA技术在疫苗研究中被证明非常有用。通过检测乙肝表面抗原（HBsAg）接种个体外周血单核细胞（PBMC）中HBsAg刺激后的IFNγ(+)分泌模式，证明了这一点。
