Efficient mRNA electroporation of peripheral blood mononuclear cells to detect memory T cell responses for immunomonitoring purposes.

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.