Laboratory of Experimental Hematology, Vaccine & Infectious Disease Institute, Faculty of Medicine, University of Antwerp, Antwerp, Belgium.
J Immunol Methods. 2010 Mar 31;354(1-2):1-10. doi: 10.1016/j.jim.2010.01.009. Epub 2010 Feb 4.
To date, the number of immunotherapy vaccines in clinical trials increases exponentially. To evaluate the efficacy of these clinical vaccination trials, we fervently need cellular immunomonitoring tools. In this study, we present a newly developed short-time assay which allows direct ex vivo analysis of multi-epitope antigen-specific T cell immune responses. This novel method is based on mRNA electroporation of isolated peripheral blood mononuclear cells (PBMC). Fresh and cryopreserved PBMC of both healthy volunteers as well as of allogeneic stem cell transplanted patients enrolled in a cytomegalovirus (CMV) dendritic cell vaccination trial were electroporated with CMV pp65-encoding mRNA. Using a direct IFN-gamma EliSPOT and intracellular cytokine flow cytometry we detected significantly higher numbers of CMV pp65-specific IFN-gamma-secreting T cells as compared to the assay with non-treated PBMC and as compared to PBMC electroporated with mRNA coding for an irrelevant protein. Compared to conventional methods to evaluate T cell-mediated immune responses, this method is time-saving and less labor-intensive because it obviates the need for in vitro cultured antigen-presenting cells and because an overnight incubation is sufficient for activation of T cells. Moreover, the use of CMV pp65-encoding mRNA will broaden the immune response because it covers every epitope with potential relevance. This is a major advantage compared to the recognition of a single epitope covered by a HLA-restricted peptides. In summary, we developed a highly efficient mRNA electroporation protocol for fresh and cryopreserved PBMC. This novel method is a rapid and elegant tool and will be convenient for monitoring the cellular immune status of patients in a clinical vaccination setting.
迄今为止，临床试验中免疫疗法疫苗的数量呈指数级增长。为了评估这些临床疫苗接种试验的疗效，我们迫切需要细胞免疫监测工具。在这项研究中，我们提出了一种新开发的短时间测定法，该方法允许直接在体外分析多表位抗原特异性 T 细胞免疫反应。这种新方法基于分离外周血单核细胞（PBMC）的 mRNA 电穿孔。我们对健康志愿者和同种异体干细胞移植患者的新鲜和冷冻 PBMC 进行 CMV pp65 编码 mRNA 的电穿孔。使用直接 IFN-γ Elispot 和细胞内细胞因子流式细胞术，我们检测到 CMV pp65 特异性 IFN-γ 分泌 T 细胞的数量明显高于未经处理的 PBMC 检测到的数量，也高于用编码无关蛋白的 mRNA 电穿孔的 PBMC 检测到的数量。与评估 T 细胞介导的免疫反应的传统方法相比，该方法省时省力，因为它不需要体外培养的抗原呈递细胞，并且仅需过夜孵育即可激活 T 细胞。此外，使用 CMV pp65 编码的 mRNA 将扩大免疫反应，因为它涵盖了每个具有潜在相关性的表位。与识别由 HLA 限制的肽覆盖的单个表位相比，这是一个主要优势。总之，我们开发了一种针对新鲜和冷冻 PBMC 的高效 mRNA 电穿孔方案。这种新方法是一种快速而优雅的工具，将方便在临床疫苗接种环境中监测患者的细胞免疫状态。
