Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination
Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination
复制标题
DOI：
10.1016/j.jim.2007.03.002
复制
发表时间：
2007-05-31
影响因子：
2.2
通讯作者：
De Rosa, Stephen C.
中科院分区：
文献类型：
--
作者：
Horton, Helen;Thomas, Evan P.;De Rosa, Stephen C.
目前正在临床试验中评估的候选 HIV-1 疫苗旨在引发 HIV-1 特异性细胞免疫。细胞内细胞因子染色 (ICS) 检测可以对抗原特异性 T 细胞进行灵敏、定量的离体评估，包括响应细胞的免疫表型分析和多种效应功能的测量。此外，使用储存的冷冻保存的 PBMC 样本使得该测定在大型功效试验中具有吸引力，因为在大型功效试验中对新鲜分离的样本进行免疫测定不太可行。在此，我们描述了优化和定量验证用于候选疫苗临床试验的 8 色 ICS 测定的广泛研究，其中包括测量产生活 IFN-γ、IL-2、TNF-α 和 IL-4 的 CD4(+) 和 CD8(+) T 细胞。我们发现，省略活性染料染色会导致对真实抗原特异性 T 细胞反应的高估高达两倍。优化后，对 8 色测定的特异性、精密度、线性、定量限和稳健性进行了验证。该测定的定量下限通常低于 0.04%，具体取决于细胞因子子集。此外，通过适当的门控，8 色测定可提供与传统 4 色测定观察到的细胞因子阳性反应相当的细胞因子阳性反应。总之，我们首次描述了定量验证的 ICS 测定，该测定允许对疫苗诱导的免疫原性进行定量和定性评估，并分析保护的免疫相关性。 (C) 2007 Elsevier B.V. 保留所有权利。
Candidate HIV-1 vaccines currently being evaluated in clinical trials are designed to elicit HIV-1-specific cellular immunity. Intracellular cytokine staining (ICS) assays allow sensitive, quantitative ex vivo assessments of antigen-specific T cells including immunophenotyping of responding cells and measurement of multiple effector functions. Additionally, the use of banked cryopreserved PBMC samples makes this assay attractive in the setting of large efficacy trials where it is less feasible to perform immunoassays on freshly isolated samples. Here we describe extensive studies to optimize and quantitatively validate the 8-color ICS assay for use in clinical trials of candidate vaccines, which includes measurement of viable IFN-gamma, IL-2, TNF-alpha and IL-4 producing CD4(+) and CD8(+) T cells. We show that omission of viability dye staining results in an over-estimate of the true antigen-specific T cell response by up to two-fold. After optimization, the 8-color assay was validated for specificity, precision, linearity, limit of quantitation and robustness. The assay has a lower quantitation limit generally below 0.04%, depending on the cytokine subset. Additionally, with appropriate gating, the 8-color assay gives comparable cytokine-positive responses to those observed with the conventional 4-color assay. In conclusion, we provide the first description of a quantitatively validated ICS assay, which permits quantitative and qualitative evaluation of vaccine-induced immunogenicity and analysis of immune correlates of protection. (C) 2007 Elsevier B.V. All rights reserved.